(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property
Organization
International Bureau
(43) International Publication Date
8 January 2004 (08.01.2004)
PCT
(10) International Publication Number
WO 2004/003019 A2
(51) International Patent Classification 7 :
C07K 16/00
(21) International Application Number:
PCT/GB2003/002804
(22) International Filing Date: 30 June 2003 (30.06.2003)
(25) Filing Language:
(26) Publication Language:
English
English
(30) Priority Data:
PCT/GB02/03014 28 June 2002 (28.06.2002) GB
0230202.4 27 December 2002 (27.12.2002) GB
(71) Applicant (for all designated States except US): DOMAN-
TIS LIMITED [GB/GB]; Granta Park, Abington, Cam-
bridgeshire CB1 6GS (GB).
(72) Inventors; and
(75) Inventors/Applicants (for US only): WINTER, Greg
[GB/GB]; MRC Laboratory of Molecular Biology, Hills
Road, Cambridge CB2 2QH (GB). TOMLINSON, Ian
[GB/GB]; Domantis Limited, Granta Park, Abington,
Cambridge CB1 6GS (GB). IGNATOVICH, Olga
[BY/GB]; Domantis Limited, Granta Park, Abington,
Cambridge CB1 6GS (GB). HOLT, Lucy [GB/GB];
Domantis Limited, Granta Park, Abington, Cambridge
CB1 6GS (GB). DE ANGELIS, Elena [IT/GB]; Domantis
Limited, Granta Park, Abington, Cambridge CB1 6GS
(GB).
(74) Agents: MASCHIO, Antonio et al.; D Young & Co, 21
New Fetter Lane, London EC4A IDA (GB).
(81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH,
GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
MX, MZ, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RU, SC,
SD, SE, SG, SK, SL, SY, TJ, TM, TN, TR, TT, TZ, UA,
UG, US, UZ, VC, VN, YU, ZA, ZM, ZW.
(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
European patent (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
ES, FI, FR, GB, GR, HU, IE, IT, LU, MC, NL, PT, RO,
SE, SI, SK, TR), OAPI patent (BF, BJ, CF, CG, CI, CM,
GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
Declaration under Rule 4.17:
— of inventorship ( Rule 4. 1 7( iv ) ) for US only
Published:
— without international search report and to be republished
upon receipt of that report
For two-letter codes and other abbreviations ; refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.
<
(54) Title: LIGAND
O
(57) Abstract: The invention provides a dual-specific ligand comprising a first immunoglobulin variable domain having a first bind-
ing specificity and a complementary or non-complementary immunoglobulin variable domain having a second binding specificity.
WO 2004/003019
PCT/GB2003/002804
Ligand
The present invention relates to dual specific ligands. In particular, the invention
provides a method for the preparation of dual-specific ligands comprising a first
5 immunoglobulin single variable domain binding to a first antigen or epitope, and a second
immunoglobulin single variable domain binding to a second antigen or epitope. More
particularly, the invention relates to dual-specific ligands wherein binding to at least one
of the first and second antigens or epitopes acts to increase the half-life of the ligand in
vivo. Open and closed conformation ligands comprising more than one binding specificity
10 are described.
Introduction
The antigen binding domain of an antibody comprises two separate regions: a heavy
15 chain variable domain (v H ) anci a light chain variable domain (v L : which can be either
V K or Vx). The antigen binding site itself is formed by six polypeptide loops: three from
V H domain (HI, H2 and H3) and three from V L domain (LI, L2 and L3). A diverse
primary repertoire of V genes that encode the v H and V L domains is produced by the
combinatorial rearrangement of gene segments. The v H § ene is produced by the
20 recombination of three gene segments, v H > D and J H- In humans, there are approximately
51 functional V H segments (Cook and Tomlinson (1995) Immunol Today, 16: 237), 25
functional D segments (Corbett et al (1997) J. Mol Biol, 268: 69) and 6 functional Jh
segments (Ravetch et al. (1981) Cell, 27: 583), depending on the haplotype. The v H
segment encodes the region of the polypeptide chain which forms the first and second
25 antigen binding loops of the v H domain (HI and H2), whilst the y H , D and J H segments
combine to form the third antigen binding loop of the y H domain (H3). The v L S ene is
produced by the recombination of only two gene segments, v L and hi humans, there
are approximately 40 functional V K segments (Schable and Zachau (1993) Biol Chem.
Hoppe-Seyler, 374: 1001), 31 functional V% segments (Williams et al (1996) J. Mol
30 Biol, 264: 220; Kawasaki et al (1997) Genome Res., 7: 250), 5 functional J K segments
(Hieter et al (1982) J. Biol Chem., 257: 1516) and 4 functional segments (Vasicek
WO 2004/003019 PCT/GB2003/002804
2
and Leder (1990) J. Exp. Med., 172: 609), depending on the haplotype. The v L segment
encodes the region of the polypeptide chain which forms the first and second antigen
binding loops of the y L domain (LI and L2), whilst the Vl an d J L segments combine to
form the third antigen binding loop of the Vl domain (L3). Antibodies selected from this
5 primary repertoire are believed to be sufficiently diverse to bind almost all antigens with
at least moderate affinity. High affinity antibodies are produced by "affinity maturation"
of the rearranged genes, in which point mutations are generated and selected by the
immune system on the basis of improved binding.
10 Analysis of the structures and sequences of antibodies has shown that five of the six
antigen binding loops (HI, H2, LI, L2, L3) possess a limited number of main-chain
conformations or canonical structures (Chothia and Lesk (1987) J. MoL Biol, 196: 901;
Chothia et al (1989) Nature, 342: 877). The main-chain conformations are determined by
(i) the length of the antigen binding loop, and (ii) particular residues, or types of residue,
15 at certain key position in the antigen binding loop and the antibody framework. Analysis
of the loop lengths and key residues has enabled us to the predict the main-chain
conformations of HI, H2, LI, L2 and L3 encoded by the majority of human antibody
sequences (Chothia et al (1992) J. MoL Biol, 227: 799; Tomlinson et al (1995) EMBO
J., 14: 4628; Williams et al (1996) J. MoL Biol, 264: 220). Although the H3 region is
20 much more diverse in terms of sequence, length and structure (due to the use of D
segments), it also forms a limited number of main-chain conformations for short loop
lengths which depend on the length and the presence of particular residues, or types of
residue, at key positions in the loop and the antibody framework (Martin et al (1996) J.
MoL Biol, 263: 800; Shirai et al (1996) FEBS Letters, 399: 1.
25
Bispecific antibodies comprising complementary pairs of Vh an d V L regions are known in
the art. These bispecific antibodies must comprise two pairs of Vh an d V L S > ea °h WVl
pair binding to a single antigen or epitope. Methods described involve hybrid hybridomas
(Milstein & Cuello AC, Nature 305:537-40), minibodies (Hu et al, (1996) Cancer Res
30 56:3055-3061;), diabodies (Holliger et al, (1993) Proc. Natl. Acad. Sci. USA 90, 6444-
6448; WO 94/13804), chelating recombinant antibodies (CRAbs; (Neri et al, (1995) J.
MoL Biol. 246, 367-373), biscFv (e.g. Atwell et al, (1996) Mol. Immunol. 33, 1301-
1312), "knobs in holes" stabilised antibodies (Carter et al, (1997) Protein Sci. 6, 781-
WO 2004/003019 PCT/GB2003/002804
3
788). In each case each antibody species comprises two antigen-binding sites, each
fashioned by a complementary pair of V H and V L domains. Each antibody is thereby able
to bind to two different antigens or epitopes at the same time, with the binding to EACH
antigen or epitope mediated by a v H and its complementary v L domain. Each of these
5 techniques presents its particular disadvantages; for instance in the case of hybrid
hybridomas, inactive V n /y L pairs can greatly reduce the fraction of bispecific IgG.
Furthermore, most bispecific approaches rely on the association of the different V H /V L
pairs or the association of v H and V L chains to recreate the two different Vi/Vl binding
sites. It is therefore impossible to control the ratio of binding sites to each antigen or
10 epitope in the assembled molecule and thus many of the assembled molecules will bind
to one antigen or epitope but not the other. In some cases it has been possible to engineer
the heavy or light chains at the sub-unit interfaces (Carter et al 9 1997) in order to improve
the number of molecules which have binding sites to both antigens or epitopes but this
never results in all molecules having binding to both antigens or epitopes.
15
There is some evidence that two different antibody binding specificities might be
incorporated into the same binding site, but these generally represent two or more
specificities that correspond to structurally related antigens or epitopes or to antibodies
that are broadly cross-reactive.. For example, cross-reactive antibodies have been
20 described, usually where the two antigens are related in sequence and structure, such as
hen egg white lysozyme and turkey lysozyme (McCafferty et al., WO 92/01047) or to
free hapten and to hapten conjugated to carrier (Griffiths AD et al. EMBO J 1994 13:14
3245-60). In a further example, WO 02/02773 (Abbott Laboratories) describes antibody
molecules with "dual specificity". The antibody molecules referred to are antibodies
25 raised or selected against multiple antigens, such that their specificity spans more than a
single antigen. Each complementary Vi/Vl P air in the antibodies of WO 02/02773
specifies a single binding specificity for two or more structurally related antigens; the v H
and v L domains in such complementary pairs do not each possess a separate specificity.
The antibodies thus have a broad single specificity which encompasses two antigens,
30 which are structurally related. Furthermore natural autoantibodies have been described
that are polyreactive (Casali & Notkins, Ann. Rev. Immunol. 7, 515-531), reacting with at
least two (usually more) different antigens or epitopes that are not structurally related. It
WO 2004/003019 PCT/GB2003/002804
4
has also been shown that selections of random peptide repertoires using phage display
technology on a monoclonal antibody will identify a range of peptide sequences that fit
the antigen binding site. Some of the sequences are highly related, fitting a consensus
sequence, whereas others are very different and have been termed mimotopes (Lane &
5 Stephen, Current Opinion in Immunology, 1993, 5, 268-271). It is therefore clear that a
natural four-chain antibody, comprising associated and complementary v H and V L
domains, has the potential to bind to many different antigens from a large universe of
known antigens. It is less clear how to create a binding site to two given antigens in the
same antibody, particularly those which are not necessarily structurally related.
10
Protein engineering methods have been suggested that may have a bearing on this. For
example it has also been proposed that a catalytic antibody could be created with a
binding activity to a metal ion through one variable domain, and to a hapten (substrate)
through contacts with the metal ion and a complementary variable domain (Barb as et al.,
15 1993 Proc. Natl. Acad. Sci USA 90, 6385-6389). However in this case, the binding and
catalysis of the substrate (first antigen) is proposed to require the binding of the metal ion
(second antigen). Thus the binding to the Vj/Vl pairing relates to a single but multi-
component antigen.
20 Methods have been described for the creation of bispecific antibodies from camel
antibody heavy chain single domains in which binding contacts for one antigen are
created in one variable domain, and for a second antigen in a second variable domain.
However the variable domains were not complementary. Thus a first heavy chain variable
domain is selected against a first antigen, and a second heavy chain variable domain
25 against a second antigen, and then both domains are linked together on the same chain to
give a bispecific antibody fragment (Conrath et al., J. Biol. Chem. 270, 27589-27594).
However the camel heavy chain single domains are unusual in that they are derived from
natural camel antibodies which have no light chains, and indeed the heavy chain single
domains are unable to associate with camel light chains to form complementary v H ^ d
30 VLP a ^ rs *
Single heavy chain variable domains have also been described, derived from natural
antibodies which are normally associated with light chains (from monoclonal antibodies
WO 2004/003019 PCT/GB2003/002804
5
or from repertoires of domains; see EP-A-0368684). These heavy chain variable
domains have been shown to interact specifically with one or more related antigens but
have not been combined with other heavy or light chain variable domains to create a
ligand with a specificity for two or more different antigens . Furthermore, these single
domains have been shown to have a very short in vivo half-life. Therefore such domains
are of limited therapeutic value.
It has been suggested to make bispecific antibody fragments by linking heavy chain
variable domains of different specificity together (as described above). The disadvantage
with this approach is that isolated antibody variable domains may have a hydrophobic
interface that normally makes interactions with the light chain and is exposed to solvent
and may be "sticky" allowing the single domain to bind to hydrophobic surfaces.
Furthermore, in the absence of a partner light chain the combination of two or more
different heavy chain variable domains and their association, possibly via their
hydrophobic interfaces, may prevent them from binding to one in not both of the ligands
they are able to bind in isolation. Moreover, in this case the heavy chain variable
domains would not be associated with complementary light chain variable domains and
thus may be less stable and readily unfold (Worn & Pluckthun, 1998 Biochemistry 37,
13120-7).
Summary of the invention
The inventors have described, in their copending international patent application WO
03/002609 as well as copending unpublished UK patent application 0230203.2, dual
specific immunoglobulin ligands which comprise immunoglobulin single variable
domains which each have different specificities. The domains may act in competition
with each other or independently to bind antigens or epitopes on target molecules.
In a first configuration, the present invention provides a further improvement in dual
specific ligands as developed by the present inventors, in which one specificity of the
ligand is directed towards a protein or polypeptide present in vivo in an organism which
can act to increase the half-life of the ligand by binding to it.
WO 2004/003019 PCT/GB2003/002804
6
Accordingly, in a first aspect, there is provided a dual-specific ligand comprising a first
immunoglobulin single variable domain having a binding specificity to a first antigen or
epitope and a second complementary immunoglobulin single variable domain having a
binding activity to a second antigen or epitope, wherein one or both of said antigens or
epitopes acts to increase the half-life of the ligand in vivo and wherein said first and
second domains lack mutually complementary domains which share the same specificity,
provided that said dual specific ligand does not consist of an anti-HSA V H domain and an
anti-P galactosidase V K domain. Preferably, that neither of the first or second variable
domains binds to human serum albumin (HSA).
Antigens or epitopes which increase the half-life of a ligand as described herein are
advantageously present on proteins or polypeptides found in an organism in vivo.
Examples include extracellular matrix proteins, blood proteins, and proteins present in
various tissues in the organism. The proteins act to reduce the rate of ligand clearance
from the blood, for example by acting as bulking agents, or by anchoring the ligand to a
desired site of action. Examples of antigens/epitopes which increase half-life in vivo are
given in Annex 1 below.
Increased half-life is useful in in vivo applications of immuno globulins, especially
antibodies and most especially antibody fragments of small size. Such fragments (Fvs,
disulphide bonded Fvs, Fabs, scFvs, dAbs) suffer from rapid clearance from the body;
thus, whilst they are able to reach most parts of the body rapidly, and are quick to produce
and easier to handle, their in vivo applications have been limited by their only brief
persistence in vivo. The invention solves this problem by providing increased half-life of
the ligands in vivo and consequently longer persistence times in the body of the functional
activity of the ligand.
Methods for pharmacokinetic analysis and determination of ligand half-life will be
familiar to those skilled in the art. Details may be found in Kenneth, A et al: Chemical
Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al
Pharmacokinetc analysis: A Practical Approach (1996). Reference is also made to
"Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2 nd Rev. ex
WO 2004/003019 PCT/GB2003/002804
7
edition (1982), which describes pharmacokinetic parameters such as t alpha and t beta
half lives and area under the curve (AUC).
Half lives (tV 2 alpha and t l A beta) and AUC can be determined from a curve of serum
concentration of ligand against time. The WinNonlin analysis package (available from
Pharsight Corp., Mountain View, CA94040, USA) can be used, for example, to model the
curve. In a first phase (the alpha phase) the ligand is undergoing mainly distribution in
the patient, with some elimination. A second phase (beta phase) is the terminal phase
when the ligand has been distributed and the serum concentration is decreasing as the
ligand is cleared from the patient. The t alpha half life is the half life of the first phase
and the t beta half life is the half life of the second phase. Thus, advantageously, the
present invention provides a ligand or a composition comprising a ligand according to the
invention having a ta half-life in the range of 15 minutes or more. In one embodiment,
the lower end of the range is 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5
hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition, or alternatively^
ligand or composition according to the invention will have a ta half life in the range of up
to and including 12 hours. In one embodiment, the upper end of the range is 11, 10, 9, 8,
7, 6 or 5 hours. An example of a suitable range is 1 to 6 hours, 2 to 5 hours or 3 to 4
hours.
Advantageously, the present invention provides a ligand or a composition comprising a
ligand according to the invention having a tp half-life in the range of 2.5 hours or more.
In one embodiment, the lower end of the range is 3 hours, 4 hours, 5 hours, 6 hours, 7
hours, 10 hours , 11 hours, or 12 hours. In addition, or alternatively, a ligand or
composition according to the invention has a tp half-life in the range of up to and
including 21 days. In one embodiment, the upper end of the range is 12 hours, 24 hours,
2 days, 3 days, 5 days, 10 days, 15 days or 20 days. Advantageously a ligand or
composition according to the invention will have a tp half life in the range 12 to 60 hours.
In a further embodiment, it will be in the range 12 to 48 hours. In a further embodiment
still, it will be in the range 12 to 26 hours.
In addition, or alternatively to the above criteria, the present invention provides a ligand
or a composition comprising a ligand according to the invention having an AUC
WO 2004/003019 PCT/GB2003/002804
8
value (area under the curve) in the range of 1 mg.min/ml or more. In one embodiment,
the lower end of the range is 5, 10, 15, 20, 30, 100, 200 or 300mg.min/ml. In addition, or
alternatively, a ligand or composition according to the invention has an AUC in the range
of up to 600 mg.min/ml. In one embodiment, the upper end of the range is 500, 400, 300,
5 200, 150, 100, 75 or 50 mg.min/ml. Advantageously a ligand according to the invention
will have a AUC in the range selected from the group consisting of the following: 1 5 to
150mg.min/ml, 15 to 100 mg.min/ml, 15 to 75 mg.min/ml, and 15 to 50mg.min/ml.
In a first embodiment, the dual specific ligand comprises two complementary variable
10 domains, i.e. two variable domains that, in their natural environment, are capable of
operating together as a cognate pair or group even if in the context of the present
invention they bind separately to their cognate epitopes. For example, the complementary
variable domains may be immunoglobulin heavy chain and light chain variable domains
(V H and V L ). V H and V L domains are advantageously provided by scFv or Fab antibody
15 fragments. Variable domains may be linked together to form multivalent ligands by, for
example: provision of a hinge region at the C-terminus of each V domain and disulphide
bonding between cysteines in the hinge regions; or provision of dAbs each with a cysteine
at the C-terminus of the domain, the cysteines being disulphide bonded together; or
production of V-CH & V-CL to produce a Fab format; or use of peptide linkers (for
20 example Gly 4 Ser linkers discussed hereinbelow) to produce dimers, trimers and further
multimers.
The inventors have found that the use of complementary variable domains allows the two
domain surfaces to pack together and be sequestered from the solvent. Furthermore the
25 complementary domains are able to stabilise each other. In addition, it allows the creation
of dual-specific IgG antibodies without the disadvantages of hybrid hybridomas as used
in the prior art, or the need to engineer heavy or light chains at the sub-unit interfaces.
The dual-specific ligands of the first aspect of the present invention have at least one
V H /V L pair. A bispecific IgG according to this invention will therefore comprise two
30 such pairs, one pair on each arm of the Y-shaped molecule. Unlike conventional
bispecific antibodies or diabodies, therefore, where the ratio of chains used is
determinative in the success of the preparation thereof and leads to practical difficulties,
the dual specific ligands of the invention are free from issues of chain balance. Chain
WO 2004/003019 PCT/GB2003/002804
9
imbalance in conventional bi-specific antibodies results from the association of two
different V L chains with two different V H chains, where V L chain 1 together with V H
chain 1 is able to bind to antigen or epitope 1 and V L chain 2 together with V H chain 2 is
able to bind to antigen or epitope 2 and the two correct pairings are in some way linked to
5 one another. Thus, only when V L chain 1 is paired with V H chain 1 and V L chain 2 is
paired with V H chain 2 in a single molecule is bi-specificity created. Such bi-specific
molecules can be created in two different ways. Firstly, they can be created by association
of two existing V H /V L pairings that each bind to a different antigen or epitope (for
example, in a bi-specific IgG). In this case the Vh/V l pairings must come all together in a
10 1:1 ratio in order to create a population of molecules all of which are bi-specific. This
never occurs (even when complementary CH domain is enhanced by "knobs into holes"
engineering) leading to a mixture of bi-specific molecules and molecules that are only
able to bind to one antigen or epitope but not the other. The second way of creating a bi-
specific antibody is by the simultaneous association of two different Vh chain with two
15 different V L chains (for example in a bi-specific diabody). In this case, although there
tends to be a preference for Vl chain 1 to pair with V H chain 1 and V L chain 2 to pair with
V H chain 2 (which can be enhanced by "knobs into holes" engineering of the V L and V H
domains), this paring is never achieved in all molecules, leading to a mixed formulation
whereby incorrect pairings occur that are unable to bind to either antigen or epitope.
20
Bi-specific antibodies constructed according to the dual-specific ligand approach
according to the first aspect of the present invention overcome all of these problems
because the binding to antigen or epitope 1 resides within the Vh or Vl domain and the
binding to antigen or epitope 2 resides with the complementary V L or V H domain,
25 respectively. Since V H and V L domains pair on a 1:1 basis all V H /V L pairings will be bi-
specific and thus all formats constructed using these V H /V L pairings (Fv, scFvs, Fabs,
minibodies, IgGs etc) will have 100% bi-specific activity.
In the context of the present invention, first and second "epitopes" are understood to be
30 epitopes which are not the same and are not bound by a single monospecific ligand. In
the first configuration of the invention, they are advantageously on different antigens, one
of which acts to increase the half-life of the ligand in vivo. Likewise, the first and second
antigens are advantageously not the same.
WO 2004/003019
10
PCT/GB2003/002804
The dual specific ligands of the invention do not include ligands as described in WO
02/02773. Thus, the ligands of the present invention do not comprise complementary
Vh/V l pairs which bind any one or more antigens or epitopes co-operatively. Instead, the
5 ligands according to the first aspect of the invention comprise a Vh/V l complementary
pair, wherein the V domains have different specificities.
Moreover, the ligands according to the first aspect of the invention comprise V h /Vl
complementary pairs having different specificities for non-structurally related epitopes or
10 antigens. Structurally related epitopes or antigens are epitopes or antigens which possess
sufficient structural similarity to be bound by a conventional V H /V L complementary pair
which acts in a co-operative manner to bind an antigen or epitope; in the case of
structurally related epitopes, the epitopes are sufficiently similar in structure that they
"fit" into the same binding pocket formed at the antigen binding site of the V H /V L dimer.
15
In a second aspect, the present invention provides a ligand comprising a first
immunoglobulin variable domain having a first antigen or epitope binding specificity and
a second immunoglobulin variable domain having a second antigen or epitope binding
specificity wherein one or both of said first and second variable domains bind to an
20 antigen which increases the half-life of the ligand in vivo, and the variable domains are
not complementary to one another.
In one embodiment, binding to one variable domain modulates the binding of the ligand
to the second variable domain.
25
In this embodiment, the variable domains may be, for example, pairs of V H domains or
pairs of Vl domains. Binding of antigen at the first site may modulate, such as enhance or
inhibit, binding of an antigen at the second site. For example, binding at the first site at
least partially inhibits binding of an antigen at a second site. In such an embodiment, the
30 ligand may for example be maintained in the body of a subject organism in vivo through
binding to a protein which increases the half-life of the ligand until such a time as it
becomes bound to the second target antigen and dissociates from the half-life increasing
protein.
WO 2004/003019
11
PCT/GB2003/002804
Modulation of binding in the above context is achieved as a consequence of the structural
proximity of the antigen binding sites relative to one another. Such structural proximity
can be achieved by the nature of the structural components linking the two or more
5 antigen binding sites, eg by the provision of a ligand with a relatively rigid structure that
holds the antigen binding sites in close proximity. Advantageously, the two or more
antigen binding sites are in physically close proximity to one another such that one site
modulates the binding of antigen at another site by a process which involves steric
hindrance and/or conformational changes within the immunoglobulin molecule.
10
The first and the second antigen binding domains may be associated either covalently or
non-covalently. In the case that the domains are covalently associated, then the
association may be mediated for example by disulphide bonds or by a polypeptide linker
such as (Gly4Ser) n? where n = from 1 to 8, eg, 2, 3, 4, 5 or 7.
15
Ligands according to the invention may be combined into non-immuno globulin multi-
ligand structures to form multivalent complexes, which bind target molecules with the
same antigen, thereby providing superior avidity, while at least one variable domain binds
an antigen to increase the half life of the multimer. For example natural bacterial
20 receptors such as SpA have been used as scaffolds for the grafting of CDRs to generate
ligands which bind specifically to one or more epitopes. Details of this procedure are
described in US 5,831,012. Other suitable scaffolds include those based on fibronectin
and affibodies. Details of suitable procedures are described in WO 98/58965. Other
suitable scaffolds include lipocallin and CTLA4, as described in van den Beuken et al, J.
25 Mol. Biol. (2001) 310, 591-601, and scaffolds such as those described in WO0069907
(Medical Research Council), which are based for example on the ring structure of
bacterial GroEL or other chaperone polypeptides.
Protein scaffolds may be combined; for example, CDRs may be grafted on to a CTLA4
30 scaffold and used together with immunoglobulin Vh or V L domains to form a ligand.
Likewise, fibronectin, lipocallin and other scaffolds maybe combined.
WO 2004/003019 PCT/GB2003/002804
12
In the case that the variable domains are selected from V-gene repertoires selected for
instance using phage display technology as herein described, then these variable domains
can comprise a universal framework region, such that is they may be recognised by a
specific generic ligand as herein defined. The use of universal frameworks, generic
5 ligands and the like is described in WO99/20749. In the present invention, reference to
phage display includes the use of both phage and/or phagemids.
/■
Where V-gene repertoires are used variation in polypeptide sequence is preferably located
within the structural loops of the variable domains. The polypeptide sequences of either
10 variable domain may be altered by DNA shuffling or by mutation in order to enhance the
interaction of each variable domain with its complementary pair.
In a preferred embodiment of the invention the 'dual-specific ligand 5 is a single chain Fv
fragment. In an alternative embodiment of the invention, the ' dual- specific ligand 5
15 consists of a Fab region of an antibody. The term "Fab region 55 includes a Fab-like
region where two VH or two VL domains are used.
The variable regions may be derived from antibodies directed against target antigens or
epitopes. Alternatively they may be derived from a repertoire of single antibody domains
20 such as those expressed on the surface of filamentous bacteriophage. Selection may be
performed as described below.
In a third aspect, the invention provides a method for producing a ligand comprising a
first immunoglobulin single variable domain having a first binding specificity and a
25 second single immunoglobulin single variable domain having a second (different) binding
specificity, one or both of the binding specificities being specific for an antigen which
increases the half-life of the ligand in vivo, the method comprising the steps of:
(a) selecting a first variable domain by its ability to bind to a first epitope,
(b) selecting a second variable region by its ability to bind to a second epitope,
30 (c) combining the variable domains; and
(d) selecting the ligand by its ability to bind to said first epitope and to said second
epitope.
WO 2004/003019 PCT/GB2003/002804
13
The ligand can bind to the first and second epitopes either simultaneously or, where there
is competition between the binding domains for epitope binding, the binding of one
domain may preclude the binding of another domain to its cognate epitope. In one
embodiment, therefore, step (d) above requires simultaneous binding to both first and
5 second (and possibly further) epitopes; in another embodiment, the binding to the first
and second epitoes is not simultaneous.
The epitopes are preferably on separate antigens.
10 Ligands advantageously comprise V h /Vl combinations, or V h /Vh or Vi/Vl combinations
of immunoglobulin variable domains, as described above. The ligands may moreover
comprise camelid Vhh domains, provided that the Vhh domain which is specific for an
antigen which increases the half-life of the ligand in vivo does not bind Hen egg white
lysozyme (HEL), porcine pancreatic alpha-amylase or NmC-A; hcg, BSA-linked RR6 azo
15 dye or S. mutans HG982 cells, as described in Conrath et ah, (2001) JBC 276:7346-7350
and W099/23221, neither of which describe the use of a specificity for an antigen which
increases half-life to increase the half life of the ligand in vivo.
In one embodiment, said first variable domain is selected for binding to said first epitope
20 in absence of a complementary variable domain. In a further embodiment, said first
variable domain is selected for binding to said first epitope/antigen in the presence of a
third variable domain in which said third variable domain is different from said second
variable domain and is complementary to the first domain. Similarly, the second domain
may be selected in the absence or presence of a complementary variable domain.
25
The antigens or epitopes targeted by the ligands of the invention, in addition to the half-
life enhancing protein, may be any antigen or epitope but advantageously is an antigen or
epitope that is targeted with therapeutic benefit. The invention provides ligands,
including open conformation, closed conformation and isolated dAb monomer ligands,
30 specific for any such target, particularly those targets further identified herein. Such
targets may be, or be part of, polypeptides, proteins or nucleic acids, which may be
naturally occurring or synthetic. In this respect, the ligand of the invention may bind the
epiotpe or antigen and act as an antagonist or agonist (eg, EPO receptor agonist). One
WO 2004/003019 PCT/GB2003/002804
14
skilled in the art will appreciate that the choice is large and varied. They may be for
instance human or animal proteins, cytokines, cytokine receptors, enzymes co-factors for
enzymes or DNA binding proteins. Suitable cytokines and growth factors include but are
not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin- 1 , EGF, EGF receptor, ENA-78,
5 Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic, fibroblast growth factor- 10,
FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-pl, insulin, IFN-y,
IGF-I, IGF-II, IL-la, IL-lp, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77
a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin a,
Inhibin P, IP- 10, keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin,
10 Mullerian inhibitory substance, monocyte colony inhibitory factor, monocyte attractant
protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-
4, MDC (67 a.a.), MDC (69 a.a.), MIG, MIP-lcx, MIP-lp, MIP-3a, MIP-3p, MIP-4,
myeloid progenitor inhibitor factor- 1 (MPIF-1), NAP -2, Neurturin, Nerve growth factor,
p-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES,
15 SDFla, SDFlp, SCF, SCGF, stem cell factor (SCF), TARC, TGF-a, TGF-P, TGF-p2,
TGF-P3, tumour necrosis factor (TNF), TNF-a, TNF-p, TNF receptor I, TNF receptor II,
TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2,
GRO/MGSA, GRO-p, GRO-y, HCC1, 1-309, HER 1, HER 2, HER 3 and HER 4.
Cytokine receptors include receptors for the foregoing cytokines. It will be appreciated
20 that this list is by no means exhaustive.
In one embodiment of the invention, the variable domains are derived from a respective
antibody directed against the antigen or epitope. In a preferred embodiment the variable
domains are derived from a repertoire of single variable antibody domains.
25
In a further aspect, the present invention provides one or more nucleic acid molecules
encoding at least a dual-specific ligand as herein defined. The dual specific ligand may
be encoded on a single nucleic acid molecule; alternatively, each domain may be encoded
by a separate nucleic acid molecule. Where the ligand is encoded by a single nucleic acid
30 molecule, the domains may be expressed as a fusion polypeptide, in the manner of a scFv
molecule, or may be separately expressed and subsequently linked together, for example
using chemical linking agents. Ligands expressed from separate nucleic acids will be
linked together by appropriate means.
WO 2004/003019
15
PCT/GB2003/002804
The nucleic acid may further encode a signal sequence for export of the polypeptides
from a host cell upon expression and may be fused with a surface component of a
filamentous bacteriophage particle (or other component of a selection display system)
5 upon expression.
In a further aspect the present invention provides a vector comprising nucleic acid
encoding a dual specific ligand according to the present invention.
10 In a yet further aspect, the present invention provides a host cell transfected with a vector
encoding a dual specific ligand according to the present invention.
Expression from such a vector may be configured to produce, for example on the surface
of a bacteriophage particle, variable domains for selection. This allows selection of
15 displayed variable regions and thus selection of 'dual-specific ligands 5 using the method
of the present invention.
The present invention further provides a kit comprising at least a dual-specific ligand
according to the present invention.
20
Dual-Specific ligands according to the present invention preferably comprise
combinations of heavy and light chain domains. For example, the dual specific ligand
may comprise a Vh domain and a Vl domain, which may be linked together in the form
of an scFv. In addition, the ligands may comprise one or more Ch or Cl domains. For
25 example, the ligands may comprise a ChI domain, C H 2 or Ch3 domain, and/or a C L
domain, Cpl, Cjli2, Cp3 or C|J,4 domains, or any combination thereof. A hinge region
domain may also be included. Such combinations of domains may, for example, mimic
natural antibodies, such as IgG or IgM, or fragments thereof, such as Fv, scFv, Fab or
F(ab') 2 molecules. Other structures, such as a single arm of an IgG molecule comprising
30 V H , V L , C h 1 and Cl domains, are envisaged.
In a preferred embodiment of the invention, the variable regions are selected from single
domain V gene repertoires. Generally the repertoire of single antibody domains is
WO 2004/003019 PCT/GB2003/002804
16
displayed on the surface of filamentous bacteriophage. In a preferred embodiment each
single antibody domain is selected by binding of a phage repertoire to antigen.
In a preferred embodiment of the invention each single variable domain may be selected
5 for binding to its target antigen or epitope in the absence of a complementary variable
region. In an alternative embodiment, the single variable domains may be selected for
binding to its target antigen or epitope in the presence of a complementary variable
region. Thus the first single variable domain may be selected in the presence of a third
complementary variable domain, and the second variable domain may be selected in the
10 presence of a fourth complementary variable domain. The complementary third or fourth
variable domain may be the natural cognate variable domain having the same specificity
as the single domain being tested, or a non-cognate complementary domain — such as a
"dummy" variable domain.
15 Preferably, the dual specific ligand of the invention comprises only two variable domains
although several such ligands may be incorporated together into the same protein, for
example two such ligands can be incorporated into an IgG or a multimeric
immunoglobulin, such as IgM. Alternatively, in another embodiment a plurality of dual
specific ligands are combined to form a multimer. For example, two different dual
20 specific ligands are combined to create a tetra-specific molecule.
It will be appreciated by one skilled in the art that the light and heavy variable regions of
a dual-specific ligand produced according to the method of the present invention may be
on the same polypeptide chain, or alternatively, on different polypeptide chains. In the
25 case that the variable regions are on different polypeptide chains, then they may be linked
via a linker, generally a flexible linker (such as a polypeptide chain), a chemical linking
group, or any other method known in the art.
In a further aspect, the present invention provides a composition comprising a dual-
30 specific ligand, obtainable by a method of the present invention, and a pharmaceutically
acceptable carrier, diluent or excipient.
WO 2004/003019 PCT/GB2003/002804
17
Moreover, the present invention provides a method for the treatment and/or prevention
of disease using a c dual-specific ligand' or a composition according to the present
invention.
5 In a second configuration, the present invention provides multispecific ligands which
comprise at least two non-complementary variable domains. For example, the ligands
may comprise a pair of V H domains or a pair of V L domains. Advantageously, the
domains are of non-camelid origin; preferably they are human domains or comprise
human framework regions (FWs) and one or more heterologous CDRs. CDRs and
10 framework regions are those regions of an immunoglobulin variable domain as defined in
the Kabat database of Sequences of Proteins of Immunological Interest.
Preferred human framework regions are those encoded by germline gene segments DP47
and DPK9. Advantageously, FW1, FW2 and FWS of a V H or V L domain have the
15 sequence of FW1, FW2 or FWS from DP47 or DPK9. The human frameworks may
optionally contain mutations, for example up to about 5 amino acid changes or up to
about 10 amino acid changes collectively in the human frameworks used in the ligands of
the invention.
20 The variable domains in the multispecific ligands according to the second configuration
of the invention may be arranged in an open or a closed conformation; that is, they may
be arranged such that the variable domains can bind their cognate ligands independently
and simultaneously, or such that only one of the variable domains may bind its cognate
ligand at any one time.
25
The inventors have realised that under certain structural conditions, non-complementary
variable domains (for example two light chain variable domains or two heavy chain
variable domains) may be present in a ligand such that binding of a first epitope to a first
variable domain inhibits the binding of a second epitope to a second variable domain,
30 even though such non-complementary domains do not operate together as a cognate pair.
WO 2004/003019 PCT/GB2003/002804
18
Advantageously, the ligand comprises two or more pairs of variable domains; that is, it
comprises at least four variable domains. Advantageously, the four variable domains
comprise frameworks of human origin.
5 In a preferred embodiment, the human frameworks are identical to those of human
germline sequences.
The present inventors consider that such antibodies will be of particular use in ligand
binding assays for therapeutic and other uses.
10
Thus, in a first aspect of the second configuration, the present invention provides a
method for producing a multispecific ligand comprising the steps of:
a) selecting a first epitope binding domain by its ability to bind to a first epitope,
b) selecting a second epitope binding domain by its ability to bind to a second
15 epitope,
c) combining the epitope binding domains; and
d) selecting the closed conformation multispecific ligand by its ability to bind to said
first second epitope and said second epitope.
20 In a further aspect of the second configuration, the invention provides method for
preparing a closed conformation multi-specific ligand comprising a first epitope binding
domain having a first epitope binding specificity and a non-complementary second
epitope binding domain having a second epitope binding specificity, wherein the first and
second binding specificities compete for epitope binding such that the closed
25 conformation multi-specific ligand may not bind both epitopes simultaneously, said
method comprising the steps of:
a) selecting a first epitope binding domain by its ability to bind to a first epitope,
b) selecting a second epitope binding domain by its ability to bind to a second
30 epitope,
c) combining the epitope binding domains such that the domains are in a closed
conformation; and
WO 2004/003019 PCT/GB2003/002804
19
d) selecting the closed conformation multispecific ligand by its ability to bind to
said first second epitope and said second epitope, but not to both said first and
second epitopes simultaneously.
5 Moreover, the invention provides a closed conformation multi-specific ligand comprising
a first epitope binding domain having a first epitope binding specificity and a non-
complementary second epitope binding domain having a second epitope binding
specificity, wherein the first and second binding specificities compete for epitope binding
such that the closed conformation multi-specific ligand may not bind both epitopes
10 simultaneously.
An alternative embodiment of the above aspect of the of the second configuration of the
invention optionally comprises a further step (bl) comprising selecting a third or further
epitope binding domain. In this way the multi-specific ligand produced, whether of open
15 or closed conformation, comprises more than two epitope binding specificities. In a
preferred aspect of the second configuration of the invention, where the multi-specific
ligand comprises more than two epitope binding domains, at least two of said domains are
in a closed conformation and compete for binding; other domains may compete for
binding or may be free to associate independently with their cognate epitope(s).
20
According to the present invention the term c multi-specific ligand' refers to a ligand
which possesses more than one epitope binding specificity as herein defined.
As herein defined the term 'closed conformation' (multi-specific ligand) means that the
25 epitope binding domains of the ligand are attached to or associated with each other,
optionally by means of a protein skeleton, such that epitope binding by one epitope
binding domain competes with epitope binding by another epitope binding domain. That
is, cognate epitopes may be bound by each epitope binding domain individually but not
simultaneosuly. The closed conformation of the ligand can be achieved using methods
30 herein described.
"Open conformation" means that the epitope binding domains of the ligand are attached
to or associated with each other, optionally by means of a protein skeleton, such that
WO 2004/003019 PCT/GB2003/002804
20
epitope binding by one epitope binding domain does not compete with epitope binding
by another epitope binding domain.
As referred to herein, the term 'competes' means that the binding of a first epitope to its
5 cognate epitope binding domain is inhibited when a second epitope is bound to its
cognate epitope binding domain. For example, binding may be inhibited sterically, for
example by physical blocking of a binding domain or by alteration of the structure or
environment of a binding domain such that its affinity or avidity for an epitope is reduced.
10 hi a further embodiment of the second configuration of the invention, the epitopes may
displace each other on binding. For example, a first epitope may be present on an antigen
which, on binding to its cognate first binding domain, causes steric hindrance of a second
binding domain, or a coformational change therein, which displaces the epitope bound to
the second binding domain.
15
Advantageously, binding is reduced by 25% or more, advantageously 40%, 50%, 60%,
70%, 80%, 90% or more, and preferably up to 100% or nearly so, such that binding is
completely inhibited. Binding of epitopes can be measured by conventional antigen
binding assays, such as ELISA, by fluorescence based techniques, including FRET, or by
20 techniques such as suface plasmon resonance which measure the mass of molecules.
According to the method of the present invention, advantageously, each epitope binding
domain is of a different epitope binding specificity.
25 In the context of the present invention, first and second "epitopes" are understood to be
epitopes which are not the same and are not bound by a single monospecific ligand. They
may be on different antigens or on the same antigen, but separated by a sufficient distance
that they do not form a single entity that could be bound by a single mono-specific V H /V L
binding pair of a conventional antibody. Experimentally, if both of the individual
30 variable domains in single chain antibody form (domain antibodies or dAbs) are
separately competed by a monospecific V H /V L ligand against two epitopes then those two
epitopes are not sufficiently far apart to be considered separate epitopes according to the
present invention.
WO 2004/003019
PCT/GB2003/002804
21
The closed conformation multispecific ligands of the invention do not include ligands as
described in WO 02/02773. Thus, the ligands of the present invention do not comprise
complementary V H /y L pairs which bind any one or more antigens or epitopes co-
5 operatively. Instead, the ligands according to the invention preferably comprise non-
complementary V H -V H or V L -V L P airs - Advantageously, each v H or V L domain in each
V H -V H or V L -V L P air has a different epitope binding specificity, and the epitope binding
sites are so arranged that the binding of an epitope at one site competes with the binding
of an epitope at another site.
10
According to the present invention, advantageously, each epitope binding domain
comprises an immunoglobulin variable domain. More advantageously, each
immunoglobulin variable domain will be either a variable light chain domain (vj or a
variable heavy chain domain V H - 111 the secon d configuration of the present invention,
15 the immunoglobulin domains when present on a ligand according to the present
invention are non-complementary, that is they do not associate to form a V n /V L antigen
binding site. Thus, multi-specific ligands as defined in the second configuration of the
invention comprise immunoglobulin domains of the same sub-type, that is either variable
light chain domains (Vl) or variable heavy chain domains (v H )- Moreover, where the
20 ligand according to the invention is in the closed conformation, the immunoglobulin
domains may be of the camelid Vhh type.
In an alternative embodiment, the ligand(s) according to the invention do not comprise a
camelid V H h domain. More particularly, the ligand(s) of the invention do not comprise
25 one or more amino acid residues that are specific to camelid V H h domains as compared to
human Vh domains.
Advantageously, the single variable domains are derived from antibodies selected for
binding activity against different antigens or epitopes. For example, the variable domains
30 may be isolated at least in part by human immunisation. Alternative methods are known
in the art, including isolation from human antibody libraries and synthesis of artificial
antibody genes.
WO 2004/003019 PCT/GB2003/002804
22
The variable domains advantageously bind super antigens, such as protein A or protein L.
Binding to superantigens is a property of correctly folded antibody variable domains, and
allows such domains to be isolated from, for example, libraries of recombinant or mutant
domains.
Epitope binding domains according to the present invention comprise a protein scaffold
and epitope interaction sites (which are advantageously on the surface of the protein
scaffold).
Epitope binding domains may also be based on protein scaffolds or skeletons other than
immunoglobulin domains. For example natural bacterial receptors such as SpA have been
used as scaffolds for the grafting of CDRs to generate ligands which bind specifically to
one or more epitopes. Details of this procedure are described in US 5,831,012. Other
suitable scaffolds include those based on fibronectin and affibodies. Details of suitable
procedures are described in WO 98/58965. Other suitable scaffolds include lipocallin and
CTLA4, as described in van den Beuken et al 9 J. Mol. Biol. (2001) 310, 591-601, and
scaffolds such as those described in WO0069907 (Medical Research Council), which are
based for example on the ring structure of bacterial GroEL or other chaperone
polypeptides.
Protein scaffolds may be combined; for example, CDRs may be grafted on to a CTLA4
scaffold and used together with immunoglobulin V H or V L domains to form a multivalent
ligand. Likewise, fibronectin, lipocallin and other scaffolds maybe combined.
25 It will be appreciated by one skilled in the art that the epitope binding domains of a closed
conformation multispecific ligand produced according to the method of the present
invention may be on the same polypeptide chain, or alternatively, on different polypeptide
chains. In the case that the variable regions are on different polypeptide chains, then they
may be linked via a linker, advantageously a flexible linker (such as a polypeptide chain),
30 a chemical linking group, or any other method known in the art.
10
15
20
WO 2004/003019 PCT/GB2003/002804
23
The first and the second epitope binding domains may be associated either covalently or
non-covalently. In the case that the domains are covalently associated, then the
association may be mediated for example by disulphide bonds.
5 In the second configuation of the invention, the first and the second epitopes are
preferably different. They may be, or be part of, polypeptides, proteins or nucleic acids,
which may be naturally occurring or synthetic. In this respect, the ligand of the invention
may bind an epiotpe or antigen and act as an antagonist or agonist (eg, EPO receptor
agonist). The epitope binding domains of the ligand in one embodiment have the same
10 epitope specificity, and may for example simultaneously bind their epitope when multiple
copies of the epitope are present on the same antigen. In another embodiment, these
epitopes are provided on different antigens such that the ligand can bind the epitopes and
bridge the antigens. One skilled in the art will appreciate that the choice of epitopes and
antigens is large and varied. They may be for instance human or animal proteins,
15 cytokines, cytokine receptors, enzymes co-factors for enzymes or DNA binding proteins.
Suitable cytokines and growth factors include but are not limited to: ApoE, Apo-SAA,
BDNF, Cardiotrophin-1, EGF, EGF receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2,
EpoR, FGF-acidic, FGF-basic, fibroblast growth factor- 10, FLT3 ligand, Fractalkine
(CX3C), GDNF, G-CSF, GM-CSF, GF-pl, insulin, IFN-y, IGF-I, IGF-H, IL-la, IL-ip,
20 IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12,
IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin a, Inhibin p, IP-10, keratinocyte growth
factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian inhibitory substance,
monocyte colony inhibitory factor, monocyte attractant protein, M-CSF, MDC (67 a.a.),
MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69
25 a.a.), MIG, MlP-la, MIP-lp, MIP-3a, MIP-3P, MIP-4, myeloid progenitor inhibitor
factor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor, p-NGF, NT-3, NT-4,
Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDFla, SDFip, SCF,
SCGF, stem cell factor (SCF), TARC, TGF-a, TGF-p, TGF-p2, TGF-p3, tumour necrosis
factor (TNF), TNF-a, TNF-p, TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF,
30 VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-p,
GRO-y, HCC1, 1-309, HER 1, HER 2, HER 3, HER 4, TACE recognition site, TNF BP-I
and TNF BP-II, as well as any target disclosed in Annex 2 or Annex 3 hereto, whether in
combination as set forth in the Annexes, in a different combination or individually.
WO 2004/003019 PCT/GB2003/002804
24
Cytokine receptors include receptors for the foregoing cytokines, e.g. IL-1 Rl; IL-6R;
IL-10R; IL-18R, as well as receptors for cytokines set forth in Annex 2 or Annex 3 and
also receptors disclosed in Annex 2 and 3. It will be appreciated that this list is by no
means exhaustive. Where the multispecific ligand binds to two epitopes (on the same or
5 different antigens), the antigen(s) may be selected from this list.
Advantageously, dual specific ligands may be used to target cytokines and other
molecules which cooperate synergistically in therapeutic situations in the body of an
organism. The invention therefore provides a method for synergising the activity of two
10 or more cytokines, comprising administering a dual specific ligand capable of binding to
said two or more cytokines. In this aspect of the invention, the dual specific ligand may
be any dual specific ligand, including a ligand composed of complementary and/or non-
complementary domains, a ligand in an open conformation, and a ligand in a closed
conformation. For example, this aspect of the invention relates to combinations of V H
15 domains and V L domains, V H domains only and V L domains only.
Synergy in a therapeutic context may be achieved in a number of ways. For example,
target combinations may be therapeutically active only if both targets are targeted by the
ligand, whereas targeting one target alone is not therapeutically effective. In another
20 embodiment, one target alone may provide some low or minimal therapeutic effect, but
together with a second target the combination provides a synergistic increase in
therapeutic effect.
Preferably, the cytokines bound by the dual specific ligands of this aspect of the invention
25 are sleeted from the list shown in Annex 2.
Moreover, dual specific ligands may be used in oncology applications, where one
specificity targets CD89, which is expressed by cytotoxic cells, and the other is tumour
specific. Examples of tumour antigens which may be targetted are given in Annex 3.
30
In one embodiment of the second configuration of the invention, the variable domains are
derived from an antibody directed against the first and/or second antigen or epitope. In a
preferred embodiment the variable domains are derived from a repertoire of single
WO 2004/003019 PCT/GB2003/002804
25
variable antibody domains. In one example, the repertoire is a repertoire that is not
created in an animal or a synthetic repertoire, hi another example, the single variable
domains are not isolated (at least in part) by animal immunisation. Thus, the single
domains can be isolated from a naive library.
5
The second configuration of the invention, in another aspect, provides a multi-specific
ligand comprising a first epitope binding domain having a first epitope binding specificity
and a non-complementary second epitope binding domain having a second epitope
binding specificity. The first and second binding specificities may be the same or
10 different.
In a further aspect, the present invention provides a closed conformation multi-specific
ligand comprising a first epitope binding domain having a first epitope binding specificity
and a non-complementary second epitope binding domain having a second epitope
15 binding specificity wherein the first and second binding specificities are capable of
competing for epitope binding such that the closed conformation multi-specific ligand
cannot bind both epitopes simultaneously.
In a still further aspect, the invention provides open conformation ligands comprising
20 non-complementary binding domains, wherein the deomains are specific for a different
epitope on the same target. Such ligands bind to targets with increased avidity.
Similarly, the invention provides multivalent ligands comprising non-complementary
binding domains specific for the same epitope and directed to targets which comprise
multiple copies of said epitope, such as IL-5, PDGF-AA, PDGF-BB, TGF beta, TGF
25 beta2, TGF beta3 and TNFa, for eample human TNF Receptor 1 and human TNFoc.
In a similar aspect, ligands according to the invention can be configured to bind
individual epitopes with low affinity, such that binding to individual epitopes is not
therapeutically significant; but the increased avidity resulting from binding to two
30 epitopes provides a theapeutic benefit. In a perticular example, epitopes may be targetted
which are present individually on normal cell types, but present together only on
abnormal or diseased cells, such as tumour cells. In such a situaton, only the abnormal or
WO 2004/003019 PCT/GB2003/002804
26
diseased cells are effectively targetted by the bispecific ligands according to the
invention.
Ligand specific for multiple copies of the same epitope, or adjacent epitopes, on the same
5 target (known as chelating dAbs) may also be trimeric or polymeric (tertrameric or more)
ligands comprising three, four or more non-complementary binding domains. For
example, ligands may be constructed comprising three or four V H domains or V L
domains.
10 Moreover, ligands are provided which bind to multisubunit targets, wherein each binding
domain is specific for a subunit of said target. The ligand may be dimeric, trimeric or
polymeric.
Preferably, the multi-specific ligands according to the above aspects of the invention are
15 obtainable by the method of the first aspect of the invention.
According to the above aspect of the second configuration of the invention,
advantageously the first epitope binding domain and the second epitope binding domains
are non-complementary immunoglobulin variable domains, as herein defined. That is
20 either V h ~Vh or V L "V L variable domains.
Chelating dAbs in particular may be prepared according to a preferred aspect of the
invention, namely the use of anchor dAbs, in which a library of dimeric, trimeric or
multimeric dAbs is constructed using a vector which comprises a constant dAb upstream
25 or downstream of a linker sequence, with a repertoire of second, third and further dAbs
being inserted on the other side of the linker. For example, the anchor or guiding dAb
maybe TAR1-5 (Vk), TAR1-27(Vk), TAR2h-5(VH) or TAR2h-6(Vic).
In alternative methodologies, the use of linkers may be avoided, for example by the use of
30 non-covalent bonding or naturall affinity between binding domains such as V H and V K .
The invention accordingly provides a method for preparing a chelating multimeric ligand
comprising the steps of:
WO 2004/003019 PCT/GB2003/002804
27
(a) providing a vector comprising a nucleic acid sequence encoding a single
binding domain specific for a first epitope on a target;
(b) providing a vector encoding a repertoire comprising second binding domains
specific for a second epitope on said target, which epitope can be the same or different to
5 the first epitope, said second epitope being adjacent to said first epitope; and
(c) expressing said first and second binding domains; and
(d) isolating those combinations of first and second binding domains which
combine together to produce a target-binding dimer.
10 The first and second epitopes are adjacent such that a multimeric ligand is capable of
binding to both epitopes simultaneously. This provides the ligand with the advantages of
increased avidity if binding. Where the epitopes are the same, the increased avidity is
obtained by the presence of multiple copies of the epitope on the target, allowing at least
two copies to be simultaneously bound in order to obtain the increased avidity effect.
15
The binding domains may be associated by several methods, as well as the use of linkers.
For example, the binding domains may comprise cys residues, avidin and streptavidin
groups or other means for non-covalent attachment post-synthesis; those combinations
which bind to the target efficiently will be isolated. Alternatively, a linker may be present
20 between the first and second binding domains, which are expressed as a single
polypeptide from a single vector, which comprises the first binding domain, the linker
and a repertoire of second binding domains, for instance as described above.
In a preferred aspect, the first and second binding domains associate naturally when
25 bound to antigen; for example, V H and V K domains, when bound to adjacent epitopes, will
naturally associate in a three-way interaction to form a stable dimer. Such associated
proteins can be isolated in a target binding assay. An advantage of this procedure is that
only binding domains which bind to closely adjacent epitopes, in the correct
conformation, will associate and thus be isolated as a result of their increased avidity for
30 the target.
In an alternative embodiment of the above aspect of the second configuration of the
invention, at least one epitope binding domain comprises a non-immunoglobulin 'protein
WO 2004/003019 PCT/GB2003/002804
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scaffold 5 or c protein skeleton 5 as herein defined. Suitable non-immuno globulin protein
scaffolds include but are not limited to any of those selected from the group consisting of:
SpA, fibronectin, GroEL and other chaperones, lipocallin, CCTLA4 and affibodies, as set
forth above.
5
According to the above aspect of the second configuration of the invention,
advantageously, the epitope binding domains are attached to a 'protein skeleton 5 .
Advantageously, a protein skeleton according to the invention is an immunoglobulin
skeleton.
10
According to the present invention, the term 'immunoglobulin skeleton 5 refers to a
protein which comprises at least one immunoglobulin fold and which acts as a nucleus for
one or more epitope binding domains, as defined herein.
15 Preferred immunoglobulin skeletons as herein defined includes any one or more of those
selected from the following: an immunoglobulin molecule comprising at least (i) the CL
(kappa or lambda subclass) domain of an antibody; or (ii) the CHI domain of an antibody
heavy chain; an immunoglobulin molecule comprising the CHI and CH2 domains of an
antibody heavy chain; an immunoglobulin molecule comprising the CHI, CH2 and CH3
20 domains of an antibody heavy chain; or any of the subset (ii) in conjunction with the CL
(kappa or lambda subclass) domain of an antibody. A hinge region domain may also be
included. Such combinations of domains may, for example, mimic natural antibodies,
such as IgG or IgM, or fragments thereof, such as Fv, scFv, Fab or F(ab 5 ) 2 molecules.
Those skilled in the art will be aware that this list is not intended to be exhaustive.
25
Linking of the skeleton to the epitope binding domains, as herein defined may be
achieved at the polypeptide level, that is after expression of the nucleic acid encoding the
skeleton and/or the epitope binding domains. Alternatively, the linking step may be
performed at the nucleic acid level. Methods of linking a protein skeleton according to the
30 present invention, to the one or more epitope binding domains include the use of protein
chemistry and/or molecular biology techniques which will be familiar to those skilled in
the art and are described herein.
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Advantageously, the closed conformation multispecific ligand may comprise a first
domain capable of binding a target molecule, and a second domain capable of binding a
molecule or group which extends the half-life of the ligand. For example, the molecule or
group may be a bulky agent, such as HSA or a cell matrix protein. As used herein, the
5 phrase "molecule or group which extends the half-life of a ligand" refers to a molecule or
chemical group which, when bound by a dual-specific ligand as described herein
increases the in vivo half-life of such dual specific ligand when administered to an
animal, relative to a ligand that does not bind that molecule or group. Examples of
molecules or groups that extend the half-life of a ligand are described hereinbelow. In a
10 preferred embodiment, the closed conformation multispecific ligand may be capable of
binding the target molecule only on displacement of the half-life enhancing molecule or
group. Thus, for example, a closed conformation multispecific ligand is maintained in
circulation in the bloodstream of a subject by a bulky molecule such as HSA. When a
target molecule is encountered, competition between the binding domains of the closed
15 conformation multispecific ligand results in displacement of the HSA and binding of the
target.
Ligands according to any aspect of the present invention, as well as dAb monomers
useful in constructing such ligands, may advantageously dissociate from their cognate
target(s) with a Kd of 300nM to 5pM (ie, 3 x 10~ 7 to 5 x 10" 12 M), preferably 50nM
to20pM, or 5nM to 200pM or InM to lOOpM, 1 x 10" 7 M or less, 1 x 10" 8 M or less, 1 x
10" 9 M or less, 1 x 10" 10 M or less, 1 x 10" 11 M or less; and/or a K off rate constant of 5 x
10" 1 to 1 x 10~ 7 S'\ preferably 1 x 10' 2 to 1 x 1(T 6 S" 1 , or 5 x 1(T 3 to 1 x 10' 5 S~\ or 5 x 10" 1
S' 1 or less, or 1 x 10" 2 S" 1 or less, or 1 x 10" 3 S" 1 or less, or 1 x 10" 4 S" 1 or less, or 1 x 10~ 5 S' 1
or less, or 1 x 10" 6 S~ l or less as determined by surface plasmon resonance. The Kd rate
constand is defined as Koff/Kon-
In particular the invention provides an anti-TNFoc dAb monomer (or dual specific ligand
comprising such a dAb), homodimer, heterodimer or homotrimer ligand, wherein each
30 dAb binds TNFa. The ligand binds to TNFce with a Kd of 300nM to 5pM (ie, 3 x 1 0' 7 to
5 x 10" 12 M), preferably 50nM to 20pM, more preferably 5nM to 200pM and most
preferably InM to lOOpM; expressed in an alternative manner, the Kd is 1 x 10' M or
less, preferably 1 x 10" 8 M or less, more preferably 1 x 10* 9 M or less, advantageously 1 x
20
25
WO 2004/003019 PCT/GB2003/002804
30
10~ 10 M or less and most preferably 1 x 10' 11 M or less; and/or a Koff rate constant of 5 x
10" 1 to 1 x 10" 7 S" 1 , preferably 1 x 10~ 2 to 1 x 1CT 6 S'\ more preferably 5 x 1CT 3 to 1 x 1CT 5
S" 1 , for example 5 x 10' 1 S' 1 or less, preferably 1 x 1(T 2 S" 1 or less, more preferably 1x10"
3 S~ l or less, advantageously 1 x 10' 4 S" 1 or less, further advantageously 1 x 10' 5 S" 1 or less,
5 and most preferably 1 x 10" 6 S" 1 or less, as detemiined by surface plasmon resonance.
Preferably, the ligand neutralises TNFa in a standard L929 assay with an ND50 of
500nM to 50pM, preferably or lOOnM to 50pM, advantageously lOnM to lOOpM, more
preferably InM to lOOpM; for example 50nM or less, preferably 5nM or less,
10 advantageously 500pM or less, more preferably 200pM or less and most preferably
lOOpM or less.
Preferably, the ligand inhibits binding of TNF alpha to TNF alpha Receptor I (p55
receptor) with an IC50 of 500nM to 50pM, preferably lOOnM to 50pM, more preferably
15 lOnM to lOOpM, advantageously InM to lOOpM; for example 50nM or less, preferably
5nM or less, more preferably 500pM or less, advantageously 200pM or less, and most
preferably lOOpM or less. Preferably, the TNFa is Human TNFa.
Furthermore, the invention provides a an anti-TNF Receptor I dAb monomer, or dual
20 specific ligand comprising such a dAb, that binds to TNF Receptor I with alQ of 300nM
to 5pM (ie, 3 x 10" 7 to 5 x 10~ 12 M), preferably 50nM to20pM, more preferably 5nM to
200pM and most preferably InM to lOOpM, for example 1 x 10" 7 M or less, preferably 1
x 10' 8 M or less, more preferably 1 x 10" 9 M or less, advantageously 1 x 10" 10 M or less
1 7
and most preferably 1 x 10" 11 M or less; and/or a Koff rate constant of 5 x 10" to 1 x 10"
25 S' 1 , preferably 1 x 1(T 2 to 1 x 10* 6 S* 1 , more preferably 5 x 1CT 3 to 1 x 10~ 5 S" 1 , for
example 5 x 10" 1 S" 1 or less, preferably 1 x 10" 2 S" 1 or less, advantageously 1 x 10" 3 S" 1 or
less, more preferably 1 x 10" 4 S" 1 or less, still more preferably 1 x 10~ 5 S" 1 or less, and most
preferably 1 x 10" 6 S" 1 or less as determined by surface plasmon resonance.
30 Preferably, the dAb monomeror ligand neutralises TNFa in a standard assay (eg, the
L929 or HeLa assays described herein) with an ND50 of 500nM to 50pM, preferably
lOOnM to 50pM, more preferably lOnM to lOOpM, advantageously InM to lOOpM; for
WO 2004/003019 PCT/GB2003/002804
31
example 50nM or less, preferably 5nM or less, more preferably 500pM or less,
advantageously 200pM or less, and most preferably lOOpM or less.
Preferably, the dAb monomer or ligand inhibits binding of TNF alpha to TNF alpha
5 Receptor I (p55 receptor) with an IC50 of 500nM to 50pM, preferably lOOnM to 50pM,
more preferably lOnM to lOOpM, advantageously InM to lOOpM; for example 50nM or
less, preferably 5nM or less, more preferably 500pM or less, advantageously 200pM or
less, and most preferably lOOpM or less. Preferably, the TNF Receptor I target is Human
TNFa.
10
Furthermore, the invention provides a dAb monomer(or dual specific ligand comprising
such a dAb) that binds to serum albumin (SA) with a Kd of InM to 500^M (ie, x 10" 9 to
5 x 10" 4 ), preferably lOOnM to 10/iM. Preferably, for a dual specific ligand comprising a
first anti-SA dAb and a second dAb to another target, the affinity (eg Kd and/or Ko ff as
15 measured by surface plasmon resonance, eg using BiaCore) of the second dAb for its
target is from 1 to 100000 times (preferably 100 to 100000, more preferably 1000 to
100000, or 10000 to 100000 times) the affinity of the first dAb for SA. For example, the
first dAb binds SA with an affinity of approximately IOjuM, while the second dAb binds
its target with an affinity of lOOpM. Preferably, the serum albumin is human serum
20 albumin (HSA).
In one embodiment, the first dAb (or a dAb monomer) binds SA (eg, HSA) with aKjof
approximately 50, preferably 70, and more preferably 100, 150 or 200 nM.
25 The invention moreover provides dimers, trimers and polymers of the aforementioned
dAb monomers, in accordance with the foregoing aspect of the present invention.
Ligands according to the invention, including dAb monomers, dimers and trimers, can be
linked to an antibody Fc region, comprising one or both of C H 2 and C H 3 domains, and
30 optionally a hinge region. For example, vectors encoding ligands linked as a single
nucleotide sequence to an Fc region may be used to prepare such polypeptides.
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In a further aspect of the second configuration of the invention, the present invention
provides one or more nucleic acid molecules encoding at least a multispecific ligand as
herein defined. In one embodiment, the ligand is a closed conformation ligand. In
another embodiment, it is an open conformation ligand. The multispecific ligand may be
5 encoded on a single nucleic acid molecule; alternatively, each epitope binding domain
may be encoded by a separate nucleic acid molecule. Where the ligand is encoded by a
single nucleic acid molecule, the domains may be expressed as a fusion polypeptide, or
may be separately expressed and subsequently linked together, for example using
chemical linking agents. Ligands expressed from separate nucleic acids will be linked
1 0 together by appropriate means.
The nucleic acid may further encode a signal sequence for export of the polypeptides
from a host cell upon expression and may be fused with a surface component of a
filamentous bacteriophage particle (or other component of a selection display system)
15 upon expression. Leader sequences, which may be used in bacterial expresion and/or
phage or phagemid display, include pelB, stll, ompA, phoA, bla and pelA.
In a further aspect of the second configuration of the invention the present invention
provides a vector comprising nucleic acid according to the present invention.
20
In a yet further aspect, the present invention provides a host cell transfected with a vector
according to the present invention.
Expression from such a vector may be configured to produce, for example on the surface
25 of a bacteriophage particle, epitope binding domains for selection. This allows selection
of displayed domains and thus selection of 'multispecific ligands' using the method of the
present invention.
In a preferred embodiment of the second configuration of the invention, the epitope
30 binding domains are immunoglobulin variable regions and are selected from single
domain V gene repertoires. Generally the repertoire of single antibody domains is
displayed on the surface of filamentous bacteriophage. In a preferred embodiment each
single antibody domain is selected by binding of a phage repertoire to antigen.
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PCT/GB2003/002804
The present invention further provides a kit comprising at least a multispecific ligand
according to the present invention, which may be an open conformation or closed
conformation ligand. Kits according to the invention may be, for example, diagnostic
5 kits, therapeutic kits, kits for the detection of chemical or biological species, and the like.
In a further aspect still of the second configuration of the invention, the present invention
provides a homogenous immunoassay using a ligand according to the present invention.
10 In a further aspect still of the second configuration of the invention, the present invention
provides a composition comprising a closed conformation multispecific ligand, obtainable
by a method of the present invention, and a pharmaceutically acceptable carrier, diluent or
excipient.
15 Moreover, the present invention provides a method for the treatment of disease using a
'closed conformation multispecific ligand' or a composition according to the present
invention.
In a preferred embodiment of the invention the disease is cancer or an inflammatory
20 disease, eg rheumatoid arthritis, asthma or Crohn's disease.
In a further aspect of the second configuration of the invention, the present invention
provides a method for the diagnosis, including diagnosis of disease using a closed
conformation multispecific ligand, or a composition according to the present invention.
25 Thus in general the binding of an analyte to a closed conformation multispecific ligand
may be exploited to displace an agent, which leads to the generation of a signal on
displacement. For example, binding of analyte (second antigen) could displace an
enzyme (first antigen) bound to the antibody providing the basis for an immunoassay,
especially if the enzyme were held to the antibody through its active site.
30
Thus in a final aspect of the second configuration, the present invention provides a
method for detecting the presence of a target molecule, comprising:
WO 2004/003019 PCT/GB2003/002804
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(a) providing a closed conformation multispecific ligand bound to an agent, said ligand
being specific for the target molecule and the agent, wherein the agent which is bound by
the ligand leads to the generation of a detectable signal on displacement from the ligand;
(b) exposing the closed conformation multispecific ligand to the target molecule; and
5 (c) detecting the signal generated as a result of the displacement of the agent.
According to the above aspect of the second configuration of the invention,
advantageously, the agent is an enzyme, which is inactive when bound by the closed
conformation multi-specific ligand. Alternatively, the agent may be any one or more
10 selected from the group consisting of the following: the substrate for an enzyme, and a
fluorescent, luminescent or chromogenic molecule which is inactive or quenched when
bound by the ligand.
Sequences similar or homologous (e.g., at least about 70% sequence identity) to the
15 sequences disclosed herein are also part of the invention. In some embodiments, the
sequence identity at the amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the sequence
identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97% 98%, 99% or higher. Alternatively, substantial identity exists when the nucleic acid
20 segments will hybridize under selective hybridization conditions (e.g., very high
stringency hybridization conditions), to the complement of the strand. The nucleic acids
may be present in whole cells, in a cell lysate, or in a partially purified or substantially
pure form.
25 Calculations of "homology" or "sequence identity" or "similarity" between two
sequences (the terms are used interchangeably herein) are performed as follows. The
sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in
one or both of a first and a second amino acid or nucleic acid sequence for optimal
alignment and non-homologous sequences can be disregarded for comparison purposes).
30 In a preferred embodiment, the length of a reference sequence aligned for comparison
purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more
preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 100% of the
length of the reference sequence. The amino acid residues or nucleotides at
WO 2004/003019 PCT/GB2003/002804
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corresponding amino acid positions or nucleotide positions are then compared. When a
position in the first sequence is occupied by the same amino acid residue or nucleotide as
the corresponding position in the second sequence, then the molecules are identical at that
position (as used herein amino acid or nucleic acid "homology" is equivalent to amino
5 acid or nucleic acid "identity"). The percent identity between the two sequences is a
function of the number of identical positions shared by the sequences, taking into account
the number of gaps, and the length of each gap, which need to be introduced for optimal
alignment of the two sequences.
10 Advantageously, the BLAST algorithm (version 2.0) is employed for sequence alignment,
with parameters set to default values. The BLAST algorithm is described in detail at the
world wide web site ("www") of the National Center for Biotechnology Information
("ncbi") of the National Institutes of Health ("nih") of the U.S. government (".gov"), in
the "/Blast/" directory, in the "blastjielp.html" file. The search parameters are defined as
15 follows, and are advantageously set to the defined default parameters.
BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm employed
by the programs blastp, blastn, blastx, tblastn, and tblastx; these programs ascribe
significance to their findings using the statistical methods of Karlin and Altschul, 1990,
20 Proc. Natl. Acad. Sci. USA 87(6):2264-8 (see the "blast Jielp.html" file, as described
above) with a few enhancements. The BLAST programs were tailored for sequence
similarity searching, for example to identify homologues to a query sequence. The
programs are not generally useful for motif-style searching. For a discussion of basic
issues in similarity searching of sequence databases, see Altschul et al. (1994).
25
The five BLAST programs available at the National Center for Biotechnology
Information web site perform the following tasks:
"blastp" compares an amino acid query sequence against a protein sequence database;
"blastn" compares a nucleotide query sequence against a nucleotide sequence database;
30 "blastx" compares the six-frame conceptual translation products of a nucleotide query
sequence (both strands) against a protein sequence database;
"tblastn" compares a protein query sequence against a nucleotide sequence database
dynamically translated in all six reading frames (both strands).
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"tblastx" compares the six-frame translations of a nucleotide query sequence against the
six-frame translations of a nucleotide sequence database.
BLAST uses the following search parameters:
HISTOGRAM Display a histogram of scores for each search; default is yes. (See
5 parameter H in the BLAST Manual).
DESCRIPTIONS Restricts the number of short descriptions of matching sequences
reported to the number specified; default limit is 100 descriptions. (See parameter V in
the manual page). See also EXPECT and CUTOFF.
ALIGNMENTS Restricts database sequences to the number specified for which high-
10 scoring segment pairs (HSPs) are reported; the default limit is 50. If more database
sequences than this happen to satisfy the statistical significance threshold for reporting
(see EXPECT and CUTOFF below), only the matches ascribed the greatest statistical
significance are reported. (See parameter B in the BLAST Manual).
EXPECT The statistical significance threshold for reporting matches against database
15 sequences; the default value is 10, such that 10 matches are expected to be found merely
by chance, according to the stochastic model of Karlin and Altschul (1990). If the
statistical significance ascribed to a match is greater than the EXPECT threshold, the
match will not be reported. Lower EXPECT thresholds are more stringent, leading to
fewer chance matches being reported. Fractional values are acceptable. (See parameter E
20 in the BLAST Manual).
CUTOFF Cutoff score for reporting high-scoring segment pairs. The default value is
calculated from the EXPECT value (see above). HSPs are reported for a database
sequence only if the statistical significance ascribed to them is at least as high as would be
ascribed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF
25 values are more stringent, leading to fewer chance matches being reported. (See
parameter S in the BLAST Manual). Typically, significance thresholds can be more
intuitively managed using EXPECT.
MATRIX Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and
TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992, Proc. Natl.
30 Aacad. Sci. USA 89(22):10915-9). The valid alternative choices include: PAM40,
PAM120, PAM250 and IDENTITY. No alternate scoring matrices are available for
BLASTN; specifying the MATRIX directive in BLASTN requests returns an error
response.
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STRAND Restrict a TBLASTN search to just the top or bottom strand of the database
sequences; or restrict a BLASTN, BLASTX or TBLASTX search to just reading frames
on the top or bottom strand of the query sequence.
FILTER Mask off segments of the query sequence that have low compositional
5 complexity, as determined by the SEG program of Wootton & Federhen (1993)
Computers and Chemistry 17:149-163, or segments consisting of short-periodicity
internal repeats, as determined by the XNU program of Claverie & States, 1993,
Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST program of
Tatusov and Lipman (see the world wide web site of the NCBI). Filtering can eliminate
10 statistically significant but biologically uninteresting reports from the blast output (e.g.,
hits against common acidic-, basic- or proline-rich regions), leaving the more biologically
interesting regions of the query sequence available for specific matching against database
sequences.
15 Low complexity sequence found by a filter program is substituted using the letter "N" in
nucleotide sequence (e.g., "N" repeated 13 times) and the letter "X" in protein sequences
(e.g., "X" repeated 9 times).
Filtering is only applied to the query sequence (or its translation products), not to
20 database sequences. Default filtering is DUST for BLASTN, SEG for other programs.
It is not unusual for nothing at all to be masked by SEG, XNU, or both, when applied to
sequences in SWISS-PROT, so filtering should not be expected to always yield an effect.
Furthermore, in some cases, sequences are masked in their entirety, indicating that the
statistical significance of any matches reported against the unfiltered query sequence
25 should be suspect.
NCBI-gi Causes NCBI gi identifiers to be shown in the output, in addition to the
accession and/or locus name.
30 Most preferably, sequence comparisons are conducted using the simple BLAST search
algorithm provided at the NCBI world wide web site described above, in the "/BLAST"
directory.
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Brief Description of the Figures
Figure 1 shows the diversification of V H /HSA at positions H50, H52, H52a, H53,
H55, H56, H58, H95, H96, H97, H98 (DVT or NNK encoded
5 respectively) which are in the antigen binding site of Vh HSA. The
sequence of Vk is diversified at positions L50, L53.
Figure 2 shows Library 1 : Germline Vk/DVT Vh,
Library 2: Germline Vk/NNK V h ,
10 Library 3: Germline V H /DVT Vk
Library 4: Germline V H /NNK V K
In phage display/ScFv format. These libraries were pre-selected for
binding to generic ligands protein A and protein L so that the majority of
the clones and selected libraries are functional. Libraries were selected on
15 HSA (first round) and jS-gal (second round) or HSA /3-gal selection or on
/3-gal (first round) and HSA (second round) /3-gal HSA selection. Soluble
scFv from these clones of PCR are amplified in the sequence. One clone
encoding a dual specific antibody K8 was chosen for further work.
20 Figure 3 shows an alignment of V H chains and V K chains.
Figure 4 shows the characterisation of the binding properties of the K8 antibody,
the binding properties of the K8 antibody characterised by monoclonal
phage ELISA, the dual specific K8 antibody was found to bind HSA and
25 /3-gal and displayed on the surface of the phage with absorbant signals
greater than 1.0. No cross reactivity with other proteins was detected.
Figure 5 shows soluble scFv ELISA performed using known concentrations of the
K8 antibody fragment. A 96-well plate was coated with 100/xg of HSA,
30 BSA and /3-gal at 10/ig/ml and 100/xg/ml of Protein A at 1/xg/ml
concentration. 50/xg of the serial dilutions of the K8 scFv was applied and
the bound antibody fragments were detected with Protein L-HRP. ELISA
results confirm the dual specific nature of the K8 antibody.
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PCT/GB2003/002804
Figure 6 shows the binding characteristics of the clone K8V K /dummy V H analysed
using soluble scFv ELISA. Production of the soluble scFv fragments was
induced by IPTG as described by Harrison et al, Methods Enzymol.
5 1996;267:83-109 and the supernatant containing scFv assayed directly.
Soluble scFv ELISA is performed as described in example 1 and the bound
scFvs were detected with Protein L-HRP. The ELISA results revealed that
this clone was still able to bind /3-gal, whereas binding BSA was abolished.
10 Figure 7 shows the sequence of variable domain vectors 1 and 2.
Figure 8 is a map of the C H vector used to construct a V H 1/V H 2 multipsecific
ligand.
1 5 Figure 9 is a map of the V K vector used to construct a V K 1/V K 2 multispecific ligand.
Figure 10 TNF receptor assay comparing TAJR1-5 dimer 4 5 TAR1-5-19 dimer 4 and
TAR1-5-19 monomer.
20 Figure 1 1 TNF receptor assay comparing TAR1-5 dimers 1-6. All dimers have been
FPLC purified and the results for the optimal dimeric species are shown.
Figure 12 TNF receptor assay of TAR1-5 19 homodimers in different formats: dAb-
linker-dAb format with 3U, 5U or 7U linker, Fab format and cysteine
25 hinge linker format.
Figure 13 Dummy VH sequence for library 1. The sequence of the VH framework
based on germline sequence DP47 - JH4b. Positions where NNK
randomisation (N=A or T or C or G nucleotides; K = G or T nucleotides)
30 has been incorporated into library 1 are indicated in bold underlined text.
Figure 14
Dummy VH sequence for library 2. The sequence of the VH framework
based on germline sequence DP47 - JH4b. Positions where NNK
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40
randomisation (N=A or T or C or G nucleotides; K = G or T nucleotides)
has been incorporated into library 2 are indicated in bold underlined text.
Figure 15 Dummy V/c sequence for library 3. The sequence of the Vk framework
based on germline sequence DP K 9 - J K l. Positions where NNK
randomisation (N=A or T or C or G nucleotides; K = G or T nucleotides)
has been incorporated into library 3 are indicated in bold underlined text.
Figure 16 Nucleotide and amino acid sequence of anti MSA dAbs MSA 16 and MSA
10 26.
Figure 17 Inhibition biacore of MSA 16 and 26. Purified dAbs MSA16 and MSA26
were analysed by inhibition biacore to determine K d . Briefly, the dAbs
were tested to determine the concentration of dAb required to achieve
15 200RUs of response on a biacore CMS chip coated with a high density of
MSA. Once the required concentrations of dAb had been determined,
MSA antigen at a range of concentrations around the expected Kd was
premixed with the dAb and incubated overnight. Binding to the MSA
coated biacore chip of dAb in each of the premixes was then measured at a
20 high flow-rate of 30 /xl/minute.
Figure 1 8 Serum levels of MSA16 following injection. Serum half life of the dAb
MSA16 was determined in mouse. MSA16 was dosed as single i.v.
injections at approx 1.5mg/kg into GDI mice. Modelling with a 2
25 compartment model showed MSA16 had a tl/2a of 0.98hr 5 a tl/2/3 of
36.5hr and an AUG of 913hr.mg/ml. MSA16 had a considerably
lengthened half life compared with HEL4 (an anti-hen egg white lysozyme
dAb) which had a tl/2a of 0.06hr and a tl/2/3 of 0.34hr.
30 Figure 19 ELISA (a) and TNF receptor assay (c) showing inhibition of TNF binding
with a Fab-like fragment comprising MSA26Ck and TAR1-5-19CH.
Addition of MSA with the Fab-like fragment reduces the level of
inhibition. An ELISA plate coated with 1 jtig/ml TNFa was probed with
WO 2004/003019 PCT/GB2003/002804
41
dual specific Vk C h and Vk Ck Fab like fragment and also with a control
TNFa binding dAb at a concentration calculated to give a similar signal on
the ELISA. Both the dual specific and control dAb were used to probe the
ELISA plate in the presence and in the absence of 2mg/ml MSA. The
5 signal in the dual specific well was reduced by more than 50% but the
signal in the dAb well was not reduced at all (see figure 19a). The same
dual specific protein was also put into the receptor assay with and without
MSA and competition by MSA was also shown (see figure 19c). This
demonstrates that binding of MSA to the dual specific is competitive with
10 binding to TNF<x
Figure 20 TNF receptor assay showing inhibiton of TNF binding with a disulphide
bonded heterodimer of TAR1-5-19 dAb and MSA16 dAb. Addition of
MSA with the dimer reduces the level of inhibiton in a dose dependant
maimer. The TNF receptor assay (figure 19 (b)) was conducted in the
presence of a constant concentration of heterodimer (18nM) and a dilution
series of MSA and HSA. The presence of HSA at a range of
concentrations (up to 2 mg/ml) did not cause a reduction in the ability of
the dimer to inhibit TNFce . However, the addition of MSA caused a dose
dependant reduction in the ability of the dimer to inhibit TNFa (figure
19a) .This demonstrates that MSA and TNFa compete for binding to the
cys bonded TAR1-5-19, MSA16 dimer. MSA and HSA alone did not
have an effect on the TNF binding level in the assay.
25
Detailed Description of the Invention
Definitions
30 Complementary Two immunoglobulin domains are "complementary" where they
belong to families of structures which form cognate pairs or groups or are derived from
such families and retain this feature. For example, a V H domain and a V L domain of an
antibody are complementary; two V H domains are not complementary, and two V L
WO 2004/003019 PCT/GB2003/002804
42
domains are not complementary. Complementary domains may be found in other
members of the immuno globulin superfamily, such as the V a and Vp (or y and S) domains
of the T-cell receptor. In the context of the second configuration of the present invention,
non-complementary domains do not bind a target molecule cooperatively, but act
5 independently on different target epitopes which may be on the same or different
molecules. Domains which are artificial, such as domains based on protein scaffolds
which do not bind epitopes unless engineered to do so, are non-complementary.
Likewise, two domains based on (for example) an immunoglobulin domain and a
fibronectin domain are not complementary.
10
Immunoglobulin This refers to a family of polypeptides which retain the
immunoglobulin fold characteristic of antibody molecules, which contains two (3 sheets
and, usually, a conserved disulphide bond. Members of the immunoglobulin superfamily
are involved in many aspects of cellular and non-cellular interactions in vivo, including
15 widespread roles in the immune system (for example, antibodies, T-cell receptor
molecules and the like), involvement in cell adhesion (for example the ICAM molecules)
and intracellular signalling (for example, receptor molecules, such as the PDGF receptor).
The present invention is applicable to all immunoglobulin superfamily molecules which
possess binding domains. Preferably, the present invention relates to antibodies.
20
Combining Variable domains according to the invention are combined to form a group
of domains; for example, complementary domains may be combined, such as V L domains
being combined with V H domains. Non-complementary domains may also be combined.
Domains may be combined in a number of ways, involving linkage of the domains by
25 covalent or non-covalent means.
Domain A domain is a folded protein structure which retains its tertiary structure
independently of the rest of the protein. Generally, domains are responsible for discrete
functional properties of proteins, and in many cases may be added, removed or
30 transferred to other proteins without loss of function of the remainder of the protein
and/or of the domain. By single antibody variable domain is meant a folded polypeptide
domain comprising sequences characteristic of antibody variable domains. It therefore
includes complete antibody variable domains and modified variable domains, for example
WO 2004/003019 PCT/GB2003/002804
43
in which one or more loops have been replaced by sequences which are not characteristic
of antibody variable domains, or antibody variable domains which have been truncated or
comprise N- or C-terminal extensions, as well as folded fragments of variable domains
which retain at least in part the binding activity and specificity of the full-length domain.
5
Repertoire A collection of diverse variants, for example polypeptide variants which
differ in their primary sequence. A library used in the present invention will encompass a
repertoire of polypeptides comprising at least 1000 members.
10 Library The term library refers to a mixture of heterogeneous polypeptides or
nucleic acids. The library is composed of members, each of which have a single
polypeptide or nucleic acid sequence. To this extent, library is synonymous with
repertoire. Sequence differences between library members are responsible for the
diversity present in the library. The library may take the form of a simple mixture of
15 polypeptides or nucleic acids, or may be in the form of organisms or cells, for example
bacteria, viruses, animal or plant cells and the like, transformed with a library of nucleic
acids. Preferably, each individual organism or cell contains only one or a limited number
of library members. Advantageously, the nucleic acids are incorporated into expression
vectors, in order to allow expression of the polypeptides encoded by the nucleic acids. In
20 a preferred aspect, therefore, a library may take the form of a population of host
organisms, each organism containing one or more copies of an expression vector
containing a single member of the library in nucleic acid form which can be expressed to
produce its corresponding polypeptide member. Thus, the population of host organisms
has the potential to encode a large repertoire of genetically diverse polypeptide variants.
25
A 'closed conformation multi-specific ligand* describes a multi-specific ligand as
herein defined comprising at least two epitope binding domains as herein defined. The
term 'closed conformation' (multi-specific ligand) means that the epitope binding
domains of the ligand are arranged such that epitope binding by one epitope binding
30 domain competes with epitope binding by another epitope binding domain. That is,
cognate epitopes may be bound by each epitope binding domain individually but not
simultaneosuly. The closed conformation of the ligand can be achieved using methods
herein described.
WO 2004/003019 PCT/GB2003/002804
44
Antibody An antibody (for example IgG, IgM, IgA, IgD or IgE) or fragment (such
as a Fab , F(ab') 2? Fv, disulphide linked Fv, scFv, closed conformation multispecific
antibody, disulphide-linked scFv, diabody) whether derived from any species naturally
producing an antibody, or created by recombinant DNA technology; whether isolated
5 from serum, B-cells, hybridomas, transfectomas, yeast or bacteria).
Dual-specific ligand A ligand comprising a first immunoglobulin single variable domain
and a second immunoglobulin single variable domain as herein defined, wherein the
variable regions are capable of binding to two different antigens or two epitopes on the
10 same antigen which are not normally bound by a monospecific immunoglobulin. For
example, the two epitopes may be on the same hapten, but are not the same epitope or
sufficiently adjacent to be bound by a monospecific ligand. The dual specific ligands
according to the invention are composed of variable domains which have different
specificities, and do not contain mutually complementary variable domain pairs which
15 have the same specificity.
Antigen A molecule that is bound by a ligand according to the present invention.
Typically, antigens are bound by antibody ligands and are capable of raising an antibody
response in vivo. It may be a polypeptide, protein, nucleic acid or other molecule.
20 Generally, the dual specific ligands according to the invention are selected for target
specificity against a particular antigen. In the case of conventional antibodies and
fragments thereof, the antibody binding site defined by the variable loops (LI, L2, L3 and
HI, H2, H3) is capable of binding to the antigen.
25 Epitope A unit of structure conventionally bound by an immunoglobulin V H /V L
pair. Epitopes define the minimum binding site for an antibody, and thus represent the
target of specificity of an antibody. In the case of a single domain antibody, an epitope
represents the unit of structure bound by a variable domain in isolation.
30 Generic ligand A ligand that binds to all members of a repertoire. Generally, not bound
through the antigen binding site as defined above. Non-limiting examples include protein
A, protein L and protein G.
WO 2004/003019 PCT/GB2003/002804
45
Selecting Derived by screening, or derived by a Darwinian selection process, in
which binding interactions are made between a domain and the antigen or epitope or
between an antibody and an antigen or epitope. Thus a first variable domain may be
selected for binding to an antigen or epitope in the presence or in the absence of a
5 complementary variable domain.
Universal framework A single antibody framework sequence corresponding to
the regions of an antibody conserved in sequence as defined by Kabat ("Sequences of
Proteins of Immunological Interest", US Department of Health and Human Services) or
10 corresponding to the human germline immunoglobulin repertoire or structure as defined
by Chothia and Lesk, (1987) J. Mol. Biol. 196:910-917. The invention provides for the
use of a single framework, or a set of such frameworks, which has been found to permit
the derivation of virtually any binding specificity though variation in the hypervariable
regions alone.
15
Half-life The time taken for the serum concentration of the ligand to reduce by 50%,
in vivo, for example due to degradation of the ligand and/or clearance or sequestration of
the ligand by natural mechanisms. The ligands of the invention are stabilised in vivo and
their half-life increased by binding to molecules which resist degradation and/or clearance
20 or sequestration. Typically, such molecules are naturally occurring proteins which
themselves have a long half-life in vivo. The half-life of a ligand is increased if its
functional activity persists, in vivo, for a longer period than a similar ligand which is not
specific for the half-life increasing molecule. Thus, a ligand specific for HSA and a target
molecule is compared with the same ligand wherein the specificity for HSA is not
25 present, that it does not bind HSA but binds another molecule. For example, it may bind
a second epitope on the target molecule. Typically, the half life is increased by 10%,
20%o, 30%, 40%, 50% or more. Increases in the range of 2x, 3x, 4x, 5x, lOx, 20x, 30x,
40x, 5 Ox or more of the half life are possible. Alternatively, or in addition, increases in
the range of up to 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx, 150x of the half life are
30 possible.
Homogeneous immunoassay An immunoassay in which analyte is detected
without need for a step of separating bound and un-bound reagents.
WO 2004/003019
46
PCT/GB2003/002804
Substantially identical (or "substantially homologous") A first amino acid or
nucleotide sequence that contains a sufficient number of identical or equivalent (e.g., with
a similar side chain, e.g., conserved amino acid substitutions) amino acid residues or
5 nucleotides to a second amino acid or nucleotide sequence such that the first and second
amino acid or nucleotide sequences have similar activities. In the case of antibodies, the
second antibody has the same binding specificity and has at least 50% of the affinity of
the same.
10 As used herein, the terms "low stringency " "medium stringency," "high stringency,"
or "very high stringency conditions" describe conditions for nucleic acid hybridization
and washing. Guidance for performing hybridization reactions can be found in Current
Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is
incorporated herein by reference in its entirety. Aqueous and nonaqueous methods are
15 described in that reference and either can be used. Specific hybridization conditions
referred to herein are as follows: (1) low stringency hybridization conditions in 6X
sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X
SSC, 0.1% SDS at least at 50°C (the temperature of the washes can be increased to 55°C
for low stringency conditions); (2) medium stringency hybridization conditions in 6X
20 SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; (3)
high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or
more washes in 0.2X SSC, 0.1% SDS at 65°C; and preferably (4) very high stringency
hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one
or more washes at 0.2X SSC, 1% SDS at 65°C. Very high stringency conditions (4) are
25 the preferred conditions and the ones that should be used unless otherwise specified.
Detailed Description of the Invention
30 Unless defined otherwise, all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture,
molecular genetics, nucleic acid chemistry, hybridisation techniques and biochemistry).
Standard techniques are used for molecular, genetic and biochemical methods (see
WO 2004/003019 PCT/GB2003/002804
47
generally, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed. (1989)
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al, Short
Protocols in Molecular Biology (1999) 4 th Ed, John Wiley & Sons, Inc. which are
incorporated herein by reference) and chemical methods.
Preparation of immunoglobulin based multi-specific ligands
Dual specific ligands according to the invention, whether open or closed in conformation
according to the desired configuration of the invention, may be prepared according to
previously established techniques, used in the field of antibody engineering, for the
preparation of scFv, "phage" antibodies and other engineered antibody molecules.
Techniques for the preparation of antibodies, and in particular bispecific antibodies, are
for example described in the following reviews and the references cited therein: Winter &
Milstein, (1991) Nature 349:293-299; Plueckthun (1992) Immunological Reviews
130:151-188; Wright et al, (1992) Crti. Rev. Immunol.l2:125-168; Holliger, P. &
Winter, G. (1993) Curr. Op. Biotechn. 4, 446-449; Carter, et al. (1995) J. Hematother. 4,
463-470; Chester, K.A. & Hawkins, R.E. (1995) Trends Biotechn. 13, 294-300;
Hoogenboom, H.R. (1997) Nature Biotechnol. 15, 125-126; Fearon, D. (1997) Nature
Biotechnol. 15, 618-619; Pliickthun, A. & Pack, P. (1997) Immunotechnology 3, 83-105;
Carter, P. & Merchant, A.M. (1997) Curr. Opin. Biotechnol. 8, 449-454; Holliger, P. &
Winter, G. (1997) Cancer Immunol, hnmunother. 45,128-130.
The invention provides for the selection of variable domains against two different
antigens or epitopes, and subsequent combination of the variable domains.
The techniques employed for selection of the variable domains employ libraries and
selection procedures which are known in the art. Natural libraries (Marks et al (1991) J.
Mol Biol, 222: 581; Vaughan et al (1996) Nature Biotech., 14: 309) which use
rearranged V genes harvested from human B cells are well known to those skilled in the
art. Synthetic libraries (Hoogenboom & Winter (1992) Mol. Biol, 227: 381; Barbas et
al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457; Nissim et al (1994) EMBO J., 13: 692;
Griffiths et al. (1994) EMBO J., 13: 3245; De Kruif et al. (1995) J. Mol. Biol, 248: 97)
are prepared by cloning immunoglobulin V genes, usually using PCR. Errors in the PCR
WO 2004/003019 PCT/GB2003/002804
48
process can lead to a high degree of randomisation. V H and/or V L libraries may be
selected against target antigens or epitopes separately, in which case single domain
binding is directly selected for, or together.
5 A preferred method for making a dual specific ligand according to the present invention
comprises using a selection system in which a repertoire of variable domains is selected
for binding to a first antigen or epitope and a repertoire of variable domains is selected for
binding to a second antigen or epitope. The selected variable first and second variable
domains are then combined and the dual-specific ligand selected for binding to both first
10 and second antigen or epitope. Closed conformation ligands are selected for binding both
first and second antigen or epitope in isolation but not simultaneously.
A. Library vector systems
15 A variety of selection systems are known in the art which are suitable for use in the
present invention. Examples of such systems are described below.
Bacteriophage lambda expression systems may be screened directly as bacteriophage
plaques or as colonies of lysogens, both as previously described (Huse et ah (1989)
20 Science, 246: 1275; Caton and Koprowski (1990) Proc. Natl. Acad. Set U.S.A., 87;
Mullinax et ah (1990) Proc. Natl Acad. Sci. U.S.A., 87: 8095; Persson et al (1991) Proc.
Natl Acad. Sci. U.S.A., 88: 2432) and are of use in the invention. Whilst such expression
systems can be used to screen up to 10 6 different members of a library, they are not really
suited to screening of larger numbers (greater than 10 6 members).
25
Of particular use in the construction of libraries are selection display systems, which
enable a nucleic acid to be linked to the polypeptide it expresses. As used herein, a
selection display system is a system that permits the selection, by suitable display means,
of the individual members of the library by binding the generic and/or target ligands.
30
Selection protocols for isolating desired members of large libraries are known in the art,
as typified by phage display techniques. Such systems, in which diverse peptide
sequences are displayed on the surface of filamentous bacteriophage (Scott and Smith
WO 2004/003019 PCT/GB2003/002804
49
(1990) Science, 249: 386), have proven useful for creating libraries of antibody
fragments (and the nucleotide sequences that encoding them) for the in vitro selection and
amplification of specific antibody fragments that bind a target antigen (McCafferty et aL,
WO 92/01047). The nucleotide sequences encoding the V H and V L regions are linked to
5 gene fragments which encode leader signals that direct them to the periplasmic space of
E. coli and as a result the resultant antibody fragments are displayed on the surface of the
bacteriophage, typically as fusions to bacteriophage coat proteins (e.g., pill or pVIII).
Alternatively, antibody fragments are displayed externally on lambda phage capsids
(phagebodies). An advantage of phage-based display systems is that, because they are
10 biological systems, selected library members can be amplified simply by growing the
phage containing the selected library member in bacterial cells. Furthermore, since the
nucleotide sequence that encode the polypeptide library member is contained on a phage
or phagemid vector, sequencing, expression and subsequent genetic manipulation is
relatively straightforward.
15
Methods for the construction of bacteriophage antibody display libraries and lambda
phage expression libraries are well known in the art (McCafferty et aL (1990) Nature,
348: 552; Kang et aL (1991) Proc. Natl Acad. Set U.S.A., 88: 4363; Clackson et aL
(1991) Nature, 352: 624; Lowman et aL (1991) Biochemistry, 30: 10832; Burton et aL
20 (1991) Proc. NatL Acad. Sci U.S.A., 88: 10134; Hoogenboom et aL (1991) Nucleic Acids
Res., 19: 4133; Chang et aL (1991) J. Immunol., 147: 3610; Breitling et aL (1991) Gene,
104: 147; Marks et al. (1991) supra; Barbas et aL (1992) supra; Hawkins and Winter
(1992) J. Immunol., 22: 867; Marks et aL, 1992, J. Biol. Chem., 267: 16007; Lerner et aL
(1992) Science, 258: 1313, incorporated herein by reference).
25
One particularly advantageous approach has been the use of scFv phage-libraries (Huston
et aL, 1988, Proc. Natl. Acad. Sci U.S.A., 85: 5879-5883; Chaudhary et al. (1990) Proc.
Natl. Acad. Sci U.S.A., 87: 1066-1070; McCafferty et aL (1990) supra; Clackson et aL
(1991) Nature, 352: 624; Marks et aL (1991) J. Mol. Biol, 222: 581; Chiswell et aL
30 (1992) Trends Biotech., 10: 80; Marks et al. (1992) J. Biol Chem., 267). Various
embodiments of scFv libraries displayed on bacteriophage coat proteins have been
described. Refinements of phage display approaches are also known, for example as
WO 2004/003019 PCT/GB2003/002804
50
described in WO96/06213 and WO92/01047 (Medical Research Council et al) and
WO97/08320 (Morphosys), which are incorporated herein by reference.
Other systems for generating libraries of polypeptides involve the use of cell-free
5 enzymatic machinery for the in vitro synthesis of the library members. In one method,
RNA molecules are selected by alternate rounds of selection against a target ligand and
PCR amplification (Tuerk and Gold (1990) Science, 249: 505; Ellington and Szostak
(1990) Nature, 346: 818). A similar technique may be used to identify DNA sequences
which bind a predetermined human transcription factor (Thiesen and Bach (1990) Nucleic
10 Acids Res., 18: 3203; Beaudry and Joyce (1992) Science, 257: 635; WO92/05258 and
W092/14843). In a similar way, in vitro translation can be used to synthesise
polypeptides as a method for generating large libraries. These methods which generally
comprise stabilised polysome complexes, are described further in WO88/08453,
WO90/05785, WO90/07003, WO91/02076, WO91/05058, and WO92/02536. Alternative
15 display systems which are not phage-based, such as those disclosed in W095/22625 and
W095/1 1922 (Affymax) use the polysomes to display polypeptides for selection.
A still further category of techniques involves the selection of repertoires in artificial
compartments, which allow the linkage of a gene with its gene product. For example, a
20 selection system in which nucleic acids encoding desirable gene products may be selected
in microcapsules formed by water-in-oil emulsions is described in WO99/02671,
WO00/40712 and Tawfik & Griffiths (1998) Nature Biotechnol 16(7), 652-6. Genetic
elements encoding a gene product having a desired activity are compartmentalised into
microcapsules and then transcribed and/or translated to produce their respective gene
25 products (RNA or protein) within the microcapsules. Genetic elements which produce
gene product having desired activity are subsequently sorted. This approach selects gene
products of interest by detecting the desired activity by a variety of means.
30 B. Library Construction .
Libraries intended for selection, may be constructed using techniques known in the art,
for example as set forth above, or may be purchased from commercial sources. Libraries
WO 2004/003019 PCT/GB2003/002804
51
which are useful in the present invention are described, for example, in WO99/20749.
Once a vector system is chosen and one or more nucleic acid sequences encoding
polypeptides of interest are cloned into the library vector, one may generate diversity
within the cloned molecules by undertaking mutagenesis prior to expression;
alternatively, the encoded proteins may be expressed and selected, as described above,
before mutagenesis and additional rounds of selection are performed. Mutagenesis of
nucleic acid sequences encoding structurally optimised polypeptides is carried out by
standard molecular methods. Of particular use is the polymerase chain reaction, or PCR,
(Mullis and Faloona (1987) Methods Enzymol, 155: 335, herein incorporated by
reference). PCR, which uses multiple cycles of DNA replication catalysed by a
thermostable, DNA-dependent DNA polymerase to amplify the target sequence of
interest, is well known in the art. The construction of various antibody libraries has been
discussed in Winter et al. (1994) Ann. Rev. Immunology 12, 433-55, and references cited
therein.
PCR is performed using template DNA (at least lfg; more usefully, 1-1000 ng) and at
least 25 pmol of oligonucleotide primers; it may be advantageous to use a larger amount
of primer when the primer pool is heavily heterogeneous, as each sequence is represented
by only a small fraction of the molecules of the pool, and amounts become limiting in the
later amplification cycles. A typical reaction mixture includes: 2ul of DNA, 25 pmol of
oligonucleotide primer, 2.5 pi of 10X PCR buffer 1 (Perkin-Elmer, Foster City, CA), 0.4
ul of 1.25 uM dNTP, 0.15 pi (or 2.5 units) of Taq DNA polymerase (Perkin Elmer,
Foster City, CA) and deionized water to a total volume of 25 pi. Mineral oil is overlaid
and the PCR is performed using a programmable thermal cycler. The length and
temperature of each step of a PCR cycle, as well as the number of cycles, is adjusted in
accordance to the stringency requirements in effect. Annealing temperature and timing
are determined both by the efficiency with which a primer is expected to anneal to a
template and the degree of mismatch that is to be tolerated; obviously, when nucleic acid
molecules are simultaneously amplified and mutagenised, mismatch is required, at least
in the first round of synthesis. The ability to optimise the stringency of primer annealing
conditions is well within the knowledge of one of moderate skill in the art. An annealing
temperature of between 30 °C and 72 °C is used. Initial denaturation of the template
molecules normally occurs at between 92°C and 99°C for 4 minutes, followed by 20-40
WO 2004/003019 PCT/GB2003/002804
52
cycles consisting of denaturation (94-99°C for 15 seconds to 1 minute), annealing
(temperature determined as discussed above; 1-2 minutes), and extension (72°C for 1-5
minutes, depending on the length of the amplified product). Final extension is generally
for 4 minutes at 72°C, and may be followed by an indefinite (0-24 hour) step at 4°C.
C. Combining single variable domains
Domains useful in the invention, once selected, may be combined by a variety of methods
known in the art, including covalent and non-covalent methods.
Preferred methods include the use of polypeptide linkers, as described, for example, in
connection with scFv molecules (Bird et al, (1988) Science 242:423-426). Discussion of
suitable linkers is provided in Bird et al. Science 242, 423-426; Hudson et al , Journal
Immunol Methods 231 (1999) 177-189; Hudson et al, Proc Nat Acad Sci USA 85, 5879-
5883. Linkers are preferably flexible, allowing the two single domains to interact. One
linker example is a (Gly 4 Ser) n linker, where n=l to 8, eg, 2, 3, 4, 5 or 7. The linkers used
in diabodies, which are less flexible, may also be employed (Holliger et al, (1993) PNAS
(USA) 90:6444-6448).
In one embodiment, the linker employed is not an immunoglobulin hinge region.
Variable domains may be combined using methods other than linkers. For example, the
use of disulphide bridges, provided through naturally-occurring or engineered cysteine
residues, may be exploited to stabilise V h -Vh>V l -Vl or V H -V L dimers (Reiter et al,
(1994) Protein Eng. 7:697-704) or by remodelling the interface between the variable
domains to improve the "fit" and thus the stability of interaction (Ridgeway et al, (1996)
Protein Eng. 7:617-621; Zhu et al, (1997) Protein Science 6:781-788).
Other techniques for joining or stabilising variable domains of immunoglobulins, and in
particular antibody V H domains, may be employed as appropriate.
In accordance with the present invention, dual specific ligands can be in "closed"
conformations in solution. A "closed" configuration is that in which the two domains (for
WO 2004/003019 PCT/GB2003/002804
53
example V H and V L ) are present in associated form, such as that of an associated V H -V L
pair which forms an antibody binding site. For example, scFv may be in a closed
conformation, depending on the arrangement of the linker used to link the V H and V L
domains. If this is sufficiently flexible to allow the domains to associate, or rigidly holds
5 them in the associated position, it is likely that the domains will adopt a closed
conformation.
Similarly, V H domain pairs and V L domain pairs may exist in a closed conformation.
Generally, this will be a function of close association of the domains, such as by a rigid
10 linker, in the ligand molecule. Ligands in a closed conformation will be unable to bind
both the molecule which increases the half-life of the ligand and a second target molecule.
Thus, the ligand will typically only bind the second target molecule on dissociation from
the molecule which increases the half-life of the ligand.
15 Moreover, the construction of V H /V H , Vi/Vl or V H /V L dimers without linkers provides
for competition between the domains.
Ligands according to the invention may moreover be in an open conformation. In such a
conformation, the ligands will be able to simultaneously bind both the molecule which
20 increases the half-life of the ligand and the second target molecule. Typically, variable
domains in an open configuration are (in the case of V H -V L pairs) held far enough apart
for the domains not to interact and form an antibody binding site and not to compete for
binding to their respective epitopes. In the case of V H /V H or Vi/V L dimers, the domains
are not forced together by rigid linkers. Naturally, such domain pairings will not compete
25 for antigen binding or form an antibody binding site.
Fab fragments and whole antibodies will exist primarily in the closed conformation,
although it will be appreciated that open and closed dual specific ligands are likely to
exist in a variety of equilibria under different circumstances. Binding of the ligand to a
30 target is likely to shift the balance of the equilibrium towards the open configuration.
Thus, certain ligands according to the invention can exist in two conformations in
solution, one of which (the open form) can bind two antigens or epitopes independently,
WO 2004/003019 PCT/GB2003/002804
54
whilst the alternative conformation (the closed form) can only bind one antigen or
epitope; antigens or epitopes thus compete for binding to the ligand in this conformation.
Although the open form of the dual specific ligand may thus exist in equilibrium with the
5 closed form in solution, it is envisaged that the equilibrium will favour the closed form;
moreover, the open form can be sequestered by target binding into a closed conformation.
Preferably, therefore, certain dual specific ligands of the invention are present in an
equilibrium between two (open and closed) conformations.
10 Dual specific ligands according to the invention may be modified in order to favour an
open or closed conformation. For example, stabilisation of V H -V L interactions with
disulphide bonds stabilises the closed conformation. Moreover, linkers used to join the
domains, including V H domain and V L domain pairs, may be constructed such that the
open from is favoured; for example, the linkers may sterically hinder the association of
15 the domains, such as by incorporation of large amino acid residues in opportune
locations, or the designing of a suitable rigid structure which will keep the domains
physically spaced apart.
D. Characterisation of the dual-specific ligand .
20
The binding of the dual-specific ligand to its specific antigens or epitopes can be tested by
methods which will be familiar to those skilled in the art and include ELISA. In a
preferred embodiment of the invention binding is tested using monoclonal phage ELISA.
25 Phage ELISA may be performed according to any suitable procedure: an exemplary
protocol is set forth below.
Populations of phage produced at each round of selection can be screened for binding by
ELISA to the selected antigen or epitope, to identify "polyclonal" phage antibodies.
30 Phage from single infected bacterial colonies from these populations can then be screened
by ELISA to identify "monoclonal" phage antibodies. It is also desirable to screen soluble
antibody fragments for binding to antigen or epitope, and this can also be undertaken by
WO 2004/003019 PCT/GB2003/002804
55
ELISA using reagents, for example, against a C- or N-terminal tag (see for example
Winter et al (1994) Ann. Rev. Immunology 12, 433-55 and references cited therein.
The diversity of the selected phage monoclonal antibodies may also be assessed by gel
5 electrophoresis of PGR products (Marks et al 1991, supra; Nissim et al 1994 supra),
probing (Tomlinson et al, 1992) J. Mol. Biol. 227, 776) or by sequencing of the vector
DNA.
E. Structure of 'Dual-specific ligands' .
As described above, an antibody is herein defined as an antibody (for example IgG, IgM,
IgA, IgA, IgE) or fragment (Fab, Fv, disulphide linked Fv, scFv, diabody) which
comprises at least one heavy and a light chain variable domain, at least two heavy chain
variable domains or at least two light chain variable domains. It may be at least partly
derived from any species naturally producing an antibody, or created by recombinant
DNA technology; whether isolated from serum, B -cells, hybridomas, transfectomas, yeast
or bacteria).
In a preferred embodiment of the invention the dual-specific ligand comprises at least one
20 single heavy chain variable domain of an antibody and one single light chain variable
domain of an antibody, or two single heavy or light chain variable domains. For example,
the ligand may comprise a V H /V L pair, a pair of V H domains or a pair of V L domains.
The first and the second variable domains of such a ligand may be on the same
25 polypeptide chain. Alternatively they may be on separate polypeptide chains. In the case
that they are on the same polypeptide chain they may be linked by a linker, which is
preferentially a peptide sequence, as described above.
The first and second variable domains may be covalently or non-covalently associated. In
30 the case that they are covalently associated, the covalent bonds may be disulphide bonds.
In the case that the variable domains are selected from V-gene repertoires selected for
instance using phage display technology as herein described, then these variable domains
10
15
WO 2004/003019 PCT/GB2003/002804
56
comprise a universal framework region, such that is they may be recognised by a specific
generic ligand as herein defined. The use of universal frameworks, generic ligands and
the like is described in WO99/20749.
5 Where V-gene repertoires are used variation in polypeptide sequence is preferably located
within the structural loops of the variable domains. The polypeptide sequences of either
variable domain may be altered by DNA shuffling or by mutation in order to enhance the
interaction of each variable domain with its complementary pair. DNA shuffling is
known in the art and taught, for example, by Stemmer, 1994, Nature 370: 389-391 and
10 U.S. Patent No. 6,297,053, both of which are incorporated herein by reference. Other
methods of mutagenesis are well known to those of skill in the art.
In a preferred embodiment of the invention the 'dual-specific ligand 5 is a single chain Fv
fragment. In an alternative embodiment of the invention, the ' dual-specific ligand'
15 consists of a Fab format.
In a further aspect, the present invention provides nucleic acid encoding at least a c dual-
specific ligand' as herein defined.
20 One skilled in the art will appreciate that, depending on the aspect of the invention, both
antigens or epitopes may bind simultaneously to the same antibody molecule.
Alternatively, they may compete for binding to the same antibody molecule. For
example, where both epitopes are bound simultaneously, both variable domains of a dual
specific ligand are able to independently bind their target epitopes. Where the domains
25 compete, the one variable domain is capable of binding its target, but not at the same time
as the other variable domain binds its cognate target; or the first variable domain is
capable of binding its target, but not at the same time as the second variable domain binds
its cognate target.
30 The variable regions may be derived from antibodies directed against target antigens or
epitopes. Alternatively they may be derived from a repertoire of single antibody domains
such as those expressed on the surface of filamentous bacteriophage. Selection may be
performed as described below.
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In general, the nucleic acid molecules and vector constructs required for the performance
of the present invention may be constructed and manipulated as set forth in standard
laboratory manuals, such as Sambrook et ah (1989) Molecular Cloning: A Laboratory
5 Manual, Cold Spring Harbor, USA.
The manipulation of nucleic acids useful in the present invention is typically carried out
in recombinant vectors.
10 Thus in a further aspect, the present invention provides a vector comprising nucleic acid
encoding at least a 'dual-specific ligand' as herein defined.
As used herein, vector refers to a discrete element that is used to introduce heterologous
DNA into cells for the expression and/or replication thereof. Methods by which to select
15 or construct and, subsequently, use such vectors are well known to one of ordinary skill in
the art. Numerous vectors are publicly available, including bacterial plasmids,
bacteriophage, artificial chromosomes and episomal vectors. Such vectors may be used
for simple cloning and mutagenesis; alternatively gene expression vector is employed. A
vector of use according to the invention may be selected to accommodate a polypeptide
20 coding sequence of a desired size, typically from 0.25 kilobase (kb) to 40 kb or more in
length A suitable host cell is transformed with the vector after in vitro cloning
manipulations. Each vector contains various functional components, which generally
include a cloning (or "polylinker") site, an origin of replication and at least one selectable
marker gene. If given vector is an expression vector, it additionally possesses one or more
25 of the following: enhancer element, promoter, transcription termination and signal
sequences, each positioned in the vicinity of the cloning site, such that they are
operatively linked to the gene encoding a ligand according to the invention.
Both cloning and expression vectors generally contain nucleic acid sequences that enable
30 the vector to replicate in one or more selected host cells. Typically in cloning vectors, this
sequence is one that enables the vector to replicate independently of the host
chromosomal DNA and includes origins of replication or autonomously replicating
sequences. Such sequences are well known for a variety of bacteria, yeast and viruses.
WO 2004/003019 PCT/GB2003/002804
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The origin of replication from the plasmid pBR322 is suitable for most Gram-negative
bacteria, the 2 micron plasmid origin is suitable for yeast, and various viral origins (e.g.
SV 40, adenovirus) are useful for cloning vectors in mammalian cells. Generally, the
origin of replication is not needed for mammalian expression vectors unless these are
5 used in mammalian cells able to replicate high levels of DNA, such as COS cells.
Advantageously, a cloning or expression vector may contain a selection gene also
referred to as selectable marker. This gene encodes a protein necessary for the survival or
growth of transformed host cells grown in a selective culture medium. Host cells not
10 transformed with the vector containing the selection gene will therefore not survive in the
culture medium. Typical selection genes encode proteins that confer resistance to
antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline,
complement auxotrophic deficiencies, or supply critical nutrients not available in the
growth media.
15
Since the replication of vectors encoding a ligand according to the present invention is
most conveniently performed in E. coli, an E. co/z-selectable marker, for example, the p-
lactamase gene that confers resistance to the antibiotic ampicillin, is of use. These can be
obtained from E. coli plasmids, such as pBR322 or a pUC plasmid such as pUC18 or
20 pUC19.
Expression vectors usually contain a promoter that is recognised by the host organism and
is operably linked to the coding sequence of interest. Such a promoter may be inducible
or constitutive. The term "operably linked" refers to a juxtaposition wherein the
25 components described are in a relationship permitting them to function in their intended
manner. A control sequence "operably linked" to a coding sequence is ligated in such a
way that expression of the coding sequence is achieved under conditions compatible with
the control sequences.
30 Promoters suitable for use with prokaryotic hosts include, for example, the (3-lactamase
and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system
and hybrid promoters such as the tac promoter. Promoters for use in bacterial systems
WO 2004/003019 PCT/GB2003/002804
59
will also generally contain a Shine-Delgarno sequence operably linked to the coding
sequence.
The preferred vectors are expression vectors that enables the expression of a nucleotide
5 sequence corresponding to a polypeptide library member. Thus, selection with the first
and/or second antigen or epitope can be performed by separate propagation and
expression of a single clone expressing the polypeptide library member or by use of any
selection display system. As described above, the preferred selection display system is
bacteriophage display. Thus, phage or phagemid vectors may be used, eg pITl or pIT2.
10 Leader sequences useful in the invention include pelB, stll, ompA, phoA, bla and pel A.
One example are phagemid vectors which have an E. colt origin of replication (for
double stranded replication) and also a phage origin of replication (for production of
single-stranded DNA). The manipulation and expression of such vectors is well known in
the art (Hoogenboom and Winter (1992) supra; Nissim et aL (1994) supra). Briefly, the
15 vector contains a P -lactamase gene to confer selectivity on the phagemid and a lac
promoter upstream of a expression cassette that consists (N to C terminal) of a pelB
leader sequence (which directs the expressed polypeptide to the periplasmic space), a
multiple cloning site (for cloning the nucleotide version of the library member),
optionally, one or more peptide tag (for detection), optionally, one or more TAG stop
20 codon and the phage protein plIL Thus, using various suppressor and non-suppressor
strains of E. coli and with the addition of glucose, iso-propyl thio-(3~D-galactoside (EPTG)
or a helper phage, such as VCS Ml 3, the vector is able to replicate as a plasmid with no
expression, produce large quantities of the polypeptide library member only or produce
phage, some of which contain at least one copy of the polypeptide-pIII fusion on their
25 surface.
Construction of vectors encoding ligands according to the invention employs
conventional ligation techniques. Isolated vectors or DNA fragments are cleaved,
tailored, and religated in the form desired to generate the required vector. If desired,
30 analysis to confirm that the correct sequences are present in the constructed vector can be
performed in a known fashion. Suitable methods for constructing expression vectors,
preparing in vitro transcripts, introducing DNA into host cells, and performing analyses
for assessing expression and function are known to those skilled in the art. The presence
WO 2004/003019 PCT/GB2003/002804
60
of a gene sequence in a sample is detected, or its amplification and/or expression
quantified by conventional methods, such as Southern or Northern analysis, Western
blotting, dot blotting of DNA, RNA or protein, in situ hybridisation,
immunocytochemistry or sequence analysis of nucleic acid or protein molecules. Those
5 skilled in the art will readily envisage how these methods may be modified, if desired.
Structure of closed conformation multispecific lisands
According to one aspect of the second configuration of the invention present invention,
10 the two or more non- complementary epitope binding domains are linked so that they are
in a closed conformation as herein defined. Advantageously, they may be further
attached to a skeleton which may, as a alternative, or on addition to a linker described
herein, facilitate the formation and/or maintenance of the closed conformation of the
epitope binding sites with respect to one another.
15
(I) Skeletons
Skeletons may be based on immunoglobulin molecules or may be non-immunoglobulin in
origin as set forth above. Preferred immunoglobulin skeletons as herein defined includes
any one or more of those selected from the following: an immunoglobulin molecule
20 comprising at least (i) the CL (kappa or lambda subclass) domain of an antibody; or (ii) the
CHI domain of an antibody heavy chain; an immunoglobulin molecule comprising the
CHI and CH2 domains of an antibody heavy chain; an immunoglobulin molecule
comprising the CHI, CH2 and CH3 domains of an antibody heavy chain; or any of the
subset (ii) in conjunction with the CL (kappa or lambda subclass) domain of an antibody.
25 A hinge region domain may also be included.. Such combinations of domains may, for
example, mimic natural antibodies, such as IgG or IgM, or fragments thereof, such as Fv,
scFv, Fab or F(ab') 2 molecules. Those skilled in the art will be aware that this list is not
intended to be exhaustive.
30 (II) Protein scaffolds
Each epitope binding domain comprises a protein scaffold and one or more CDRs which
are involved in the specific interaction of the domain with one or more epitopes.
Advantageously, an epitope binding domain according to the present invention comprises
WO 2004/003019 PCT/GB2003/002804
61
three CDRs. Suitable protein scaffolds include any of those selected from the group
consisting of the following: those based on immunoglobulin domains, those based on
fibronectin, those based on affibodies, those based on CTLA4, those based on chaperones
such as GroEL, those based on lipocallin and those based on the bacterial Fc receptors
5 SpA and SpD. Those skilled in the art will appreciate that this list is not intended to be
exhaustive.
F: Scaffolds for use in Constructing Dual Specific Ligands
10 i. Selection of the main-chain conformation
The members of the immunoglobulin superfamily all share a similar fold for their
polypeptide chain. For example, although antibodies are highly diverse in terms of their
primary sequence, comparison of sequences and cryst alio graphic structures has revealed
that, contrary to expectation, five of the six antigen binding loops of antibodies (HI, H2,
15 LI, L2, L3) adopt a limited number of main-chain conformations, or canonical structures
(Chothia and Lesk (1987) J. Mol Biol, 196: 901; Chothia et al (1989) Nature, 342: 877).
Analysis of loop lengths and key residues has therefore enabled prediction of the main-
chain conformations of HI, H2, LI, L2 and L3 found in the majority of human antibodies
(Chothia et al (1992) J. Mol Biol, 227: 799; Tomlinson et ah (1995) EMBO J., 14:
20 4628; Williams et al (1996) J. Moh Biol, 264: 220). Although the H3 region is much
more diverse in terms of sequence, length and structure (due to the use of D segments), it
also forms a limited number of main-chain conformations for short loop lengths which
depend on the length and the presence of particular residues, or types of residue, at key
positions in the loop and the antibody framework (Martin et al (1996) J. Mol Biol, 263:
25 800; Shirai et al (1996) FEBS Letters, 399: 1).
The dual specific ligands of the present invention are advantageously assembled from
libraries of domains, such as libraries of Vh domains and/or libraries of Vl domains.
Moreover, the dual specific ligands of the invention may themselves be provided in the
30 form of libraries. In one aspect of the present invention, libraries of dual specific ligands
and/or domains are designed in which certain loop lengths and key residues have been
chosen to ensure that the main-chain conformation of the members is known.
Advantageously, these are real conformations of immunoglobulin superfamily molecules
WO 2004/003019 PCT/GB2003/002804
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found in nature, to minimise the chances that they are non-functional, as discussed
above. Germline V gene segments serve as one suitable basic framework for constructing
antibody or T-cell receptor libraries; other sequences are also of use. Variations may
occur at a low frequency, such that a small number of functional members may possess an
5 altered main-chain conformation, which does not affect its function.
Canonical structure theory is also of use to assess the number of different main-chain
conformations encoded by ligands, to predict the main-chain conformation based on
ligand sequences and to chose residues for diversification which do not affect the
10 canonical structure. It is known that, in the human V K domain, the LI loop can adopt one
of four canonical structures, the L2 loop has a single canonical structure and that 90% of
human V K domains adopt one of four or five canonical structures for the L3 loop
(Tomlinson et aL (1995) supra); thus, in the V K domain alone, different canonical
structures can combine to create a range of different main-chain conformations. Given
15 that the domain encodes a different range of canonical structures for the LI, L2 and L3
loops and that V K and V\ domains can pair with any Vh domain which can encode several
canonical structures for the HI and H2 loops, the number of canonical structure
combinations observed for these five loops is very large. This implies that the generation
of diversity in the main-chain conformation may be essential for the production of a wide
20 range of binding specificities. However, by constructing an antibody library based on a
single known main-chain conformation it has been found, contrary to expectation, that
diversity in the main-chain conformation is not required to generate sufficient diversity to
target substantially all antigens. Even more surprisingly, the single main-chain
conformation need not be a consensus structure - a single naturally occurring
25 conformation can be used as the basis for an entire library. Thus, in a preferred aspect, the
dual-specific ligands of the invention possess a single known main-chain conformation.
The single main-chain conformation that is chosen is preferably commonplace among
molecules of the immunoglobulin superfamily type in question. A conformation is
30 commonplace when a significant number of naturally occurring molecules are observed
to adopt it. Accordingly, in a preferred aspect of the invention, the natural occurrence of
the different main-chain conformations for each binding loop of an immunoglobulin
domain are considered separately and then a naturally occurring variable domain is
WO 2004/003019 PCT/GB2003/002804
63
chosen which possesses the desired combination of main-chain conformations for the
different loops. If none is available, the nearest equivalent may be chosen. It is preferable
that the desired combination of main-chain conformations for the different loops is
created by selecting germline gene segments which encode the desired main-chain
5 conformations. It is more preferable, that the selected germline gene segments are
frequently expressed in nature, and most preferable that they are the most frequently
expressed of all natural germline gene segments.
In designing dual specific ligands or libraries thereof the incidence of the different main-
10 chain conformations for each of the six antigen binding loops may be considered
separately. For HI, H2, LI, L2 and L3, a given conformation that is adopted by between
20% and 100% of the antigen binding loops of naturally occurring molecules is chosen.
Typically, its observed incidence is above 35% (i.e. between 35% and 100%) and, ideally,
above 50% or even above 65%. Since the vast majority of H3 loops do not have canonical
15 structures, it is preferable to select a main-chain conformation which is commonplace
among those loops which do display canonical structures. For each of the loops, the
conformation which is observed most often in the natural repertoire is therefore selected.
In human antibodies, the most popular canonical structures (CS) for each loop are as
follows: HI - CS 1 (79% of the expressed repertoire), H2 - CS 3 (46%), LI - CS 2 of
20 V K (39%), L2 - CS 1 (100%), L3 - CS 1 of V K (36%) (calculation assumes a k:A, ratio of
70:30, Hood et al. (1967) Cold Spring Harbor Symp. Quant BioL, 48: 133). For H3 loops
that have canonical structures, a CDR3 length (Kabat et al. (1991) Sequences of proteins
of immunological interest, U.S. Department of Health and Human Services) of seven
residues with a salt-bridge from residue 94 to residue 101 appears to be the most
25 common. There are at least 16 human antibody sequences in the EMBL data library with
the required H3 length and key residues to form this conformation and at least two
crystallographic structures in the protein data bank which can be used as a basis for
antibody modelling (2cgr and ltet). The most frequently expressed germline gene
segments that this combination of canonical structures are the Vh segment 3-23 (DP-47),
30 the J H segment JH4b, the V K segment 02/012 (DPK9) and the J K segment J K 1. V H
segments DP45 and DP38 are also suitable. These segments can therefore be used in
combination as a basis to construct a library with the desired single main-chain
conformation.
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PCT/GB2003/002804
Alternatively, instead of choosing the single main-chain conformation based on the
natural occurrence of the different main-chain conformations for each of the binding
loops in isolation, the natural occurrence of combinations of main-chain conformations is
used as the basis for choosing the single main-chain conformation. In the case of
antibodies, for example, the natural occurrence of canonical structure combinations for
any two, three, four, five or for all six of the antigen binding loops can be determined.
Here, it is preferable that the chosen conformation is commonplace in naturally occurring
antibodies and most preferable that it observed most frequently in the natural repertoire.
Thus, in human antibodies, for example, when natural combinations of the five antigen
binding loops, HI, H2, LI, L2 and L3, are considered, the most frequent combination of
canonical structures is determined and then combined with the most popular
conformation for the H3 loop, as a basis for choosing the single main-chain conformation.
ii. Diversification of the canonical sequence
Having selected several known main-chain conformations or, preferably a single
known main-chain conformation, dual specific ligands according to the invention or
libraries for use in the invention can be constructed by varying the binding site of the
molecule in order to generate a repertoire with structural and/or functional diversity. This
means that variants are generated such that they possess sufficient diversity in their
structure and/or in their function so that they are capable of providing a range of
activities.
The desired diversity is typically generated by varying the selected molecule at one or
more positions. The positions to be changed can be chosen at random or are preferably
selected. The variation can then be achieved either by randomisation, during which the
resident amino acid is replaced by any amino acid or analogue thereof, natural or
synthetic, producing a very large number of variants or by replacing the resident amino
acid with one or more of a defined subset of amino acids, producing a more limited
number of variants.
Various methods have been reported for introducing such diversity. Error-prone PCR
(Hawkins et al (1992) J. Mol Biol., 226: 889), chemical mutagenesis (Deng et ah (1994)
WO 2004/003019 PCT/GB2003/002804
65
J. Biol Chem., 269: 9533) or bacterial mutator strains (Low et al (1996) X Mol Biol,
260: 359) can be used to introduce random mutations into the genes that encode the
molecule. Methods for mutating selected positions are also well known in the art and
include the use of mismatched oligonucleotides or degenerate oligonucleotides, with or
5 without the use of PGR. For example, several synthetic antibody libraries have been
created by targeting mutations to the antigen binding loops. The H3 region of a human
tetanus toxoid-binding Fab has been randomised to create a range of new binding
specificities (Barbas et al (1992) Proc. Natl Acad. Set USA, 89: 4457). Random or
semi-random H3 and L3 regions have been appended to germline V gene segments to
10 produce large libraries with unmutated framework regions (Hoogenboom & Winter
(1992) J. Mol Biol, 227: 381; Barbas et al (1992) Proc. Natl Acad. Set USA, 89: 4457;
Nissim et al (1994) EMBO J., 13: 692; Griffiths et al (1994) EMBO J., 13: 3245; De
Kraif et al (1995) J. Mol. Biol, 248: 97). Such diversification has been extended to
include some or all of the other antigen binding loops (Crameri et al (1996) Nature Med.,
15 2: 100; Riechmann et al (1995) Bio/Technology, 13: 475; Morphosys, WO97/08320,
supra).
Since loop randomisation has the potential to create approximately more than 10 15
structures for H3 alone and a similarly large number of variants for the other five loops, it
20 is not feasible using current transformation technology or even by using cell free systems
to produce a library representing all possible combinations. For example, in one of the
in
largest libraries constructed to date, 6 x 10 different antibodies, which is only a fraction
of the potential diversity for a library of this design, were generated (Griffiths et al.
(1994) supra).
25
In a preferred embodiment, only those residues which are directly involved in creating or
modifying the desired function of the molecule are diversified. For many molecules, the
function will be to bind a target and therefore diversity should be concentrated in the
target binding site, while avoiding changing residues which are crucial to the overall
30 packing of the molecule or to maintaining the chosen main-chain conformation.
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Diversification of the canonical sequence as it applies to antibody domains
In the case of antibody dual-specific ligands, the binding site for the target is most
often the antigen binding site. Thus, in a highly preferred aspect, the invention provides
libraries of or for the assembly of antibody dual-specific ligands in which only those
5 residues in the antigen binding site are varied. These residues are extremely diverse in the
human antibody repertoire and are known to make contacts in high-resolution
antibody/antigen complexes. For example, in L2 it is known that positions 50 and 53 are
diverse in naturally occurring antibodies and are observed to make contact with the
antigen. In contrast, the conventional approach would have been to diversify all the
10 residues in the corresponding Complementarity Determining Region (CDR1) as defined
by Kabat et al. (1991, supra), some seven residues compared to the two diversified in the
library for use according to the invention. This represents a significant improvement in
terms of the functional diversity required to create a range of antigen binding specificities.
15 In nature, antibody diversity is the result of two processes: somatic recombination of
germline V, D and J gene segments to create a naive primary repertoire (so called
germline and junctional diversity) and somatic hypermutation of the resulting rearranged
V genes. Analysis of human antibody sequences has shown that diversity in the primary
repertoire is focused at the centre of the antigen binding site whereas somatic
20 hypermutation spreads diversity to regions at the periphery of the antigen binding site that
are highly conserved in the primary repertoire (see Tomlinson et al. (1996) J. Mol. Biol,
256: 813). This complementarity has probably evolved as an efficient strategy for
searching sequence space and, although apparently unique to antibodies, it can easily be
applied to other polypeptide repertoires. The residues which are varied are a subset of
25 those that form the binding site for the target. Different (including overlapping) subsets of
residues in the target binding site are diversified at different stages during selection, if
desired.
In the case of an antibody repertoire, an initial 'naive' repertoire is created where some,
30 but not all, of the residues in the antigen binding site are diversified. As used herein in
this context, the term "naive" refers to antibody molecules that have no pre-determined
target. These molecules resemble those which are encoded by the immunoglobulin genes
of an individual who has not undergone immune diversification, as is the case with fetal
WO 2004/003019 PCT/GB2003/002804
67
and newborn individuals, whose immune systems have not yet been challenged by a
wide variety of antigenic stimuli. This repertoire is then selected against a range of
antigens or epitopes. If required, further diversity can then be introduced outside the
region diversified in the initial repertoire. This matured repertoire can be selected for
5 modified function, specificity or affinity.
The invention provides two different naive repertoires of binding domains for the
construction of dual specific ligands, or a naive library of dual specific ligands, in which
some or all of the residues in the antigen binding site are varied. The "primary" library
10 mimics the natural primary repertoire, with diversity restricted to residues at the centre of
the antigen binding site that are diverse in the germline V gene segments (germline
diversity) or diversified during the recombination process (junctional diversity). Those
residues which are diversified include, but are not limited to, H50, H52, H52a, H53, H55,
H56, H58, H95, H96, H97, H98, L50, L53, L91, L92, L93, L94 and L96. In the "somatic"
15 library, diversity is restricted to residues that are diversified during the recombination
process (junctional diversity) or are highly somatically mutated). Those residues which
are diversified include, but are not limited to: H31, H33, H35, H95, H96, H97, H98, L30,
L31, L32, L34 and L96. All the residues listed above as suitable for diversification in
these libraries are known to make contacts in one or more antibody-antigen complexes.
20 Since in both libraries, not all of the residues in the antigen binding site are varied,
additional diversity is incorporated during selection by varying the remaining residues, if
it is desired to do so. It shall be apparent to one skilled in the art that any subset of any of
these residues (or additional residues which comprise the antigen binding site) can be
used for the initial and/or subsequent diversification of the antigen binding site.
25
In the construction of libraries for use in the invention, diversification of chosen positions
is typically achieved at the nucleic acid level, by altering the coding sequence which
specifies the sequence of the polypeptide such that a number of possible amino acids (all
20 or a subset thereof) can be incorporated at that position. Using the IUPAC
30 nomenclature, the most versatile codon is NNK, which encodes all amino acids as well as
the TAG stop codon. The NNK codon is preferably used in order to introduce the
required diversity. Other codons which achieve the same ends are also of use, including
WO 2004/003019 PCT/GB2003/002804
68
the NNN codon, which leads to the production of the additional stop codons TGA and
TAA.
A feature of side-chain diversity in the antigen binding site of human antibodies is a
5 pronounced bias which favours certain amino acid residues. If the amino acid
composition of the ten most diverse positions in each of the V H , V K and V x regions are
summed, more than 76% of the side-chain diversity comes from only seven different
residues, these being, serine (24%), tyrosine (14%), asparagine (11%), glycine (9%),
alanine (7%), aspartate (6%) and threonine (6%). This bias towards hydrophilic residues
10 and small residues which can provide main-chain flexibility probably reflects the
evolution of surfaces which are predisposed to binding a wide range of antigens or
epitopes and may help to explain the required promiscuity of antibodies in the primary
repertoire.
15 Since it is preferable to mimic this distribution of amino acids, the distribution of amino
acids at the positions to be varied preferably mimics that seen in the antigen binding site
of antibodies. Such bias in the substitution of amino acids that permits selection of certain
polypeptides (not just antibody polypeptides) against a range of target antigens is easily
applied to any polypeptide repertoire. There are various methods for biasing the amino
20 acid distribution at the position to be varied (including the use of tri-nucleotide
mutagenesis, see WO97/08320), of which the preferred method, due to ease of synthesis,
is the use of conventional degenerate codons. By comparing the amino acid profile
encoded by all combinations of degenerate codons (with single, double, triple and
quadruple degeneracy in equal ratios at each position) with the natural amino acid use it is
25 possible to calculate the most representative codon. The codons (AGT)(AGC)T,
(AGT)(AGC)C and (AGT)(AGC)(CT) - that is, DVT, DVC and DVY, respectively using
RJPAC nomenclature - are those closest to the desired amino acid profile: they encode
22% serine and 11% tyrosine, asparagine, glycine, alanine, aspartate, threonine and
cysteine. Preferably, therefore, libraries are constructed using either the DVT, DVC or
30 DVY codon at each of the diversified positions.
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G: Antigens capable of increasing ligand half-life
The dual specific ligands according to the invention, in one configuration thereof, are
capable of binding to one or more molecules which can increase the half-life of the ligand
5 in vivo. Typically, such molecules are polypeptides which occur naturally in vivo and
which resist degradation or removal by endogenous mechanisms which remove unwanted
material from the organism. For example, the molecule which increases the half-life of
the organism may be selected from the following:
10 Proteins from the extracellular matrix; for example collagen, laminins, integrins and
fibronectin. Collagens are the major proteins of the extracellular matrix. About 15 types
of collagen molecules are currently known, found in different parts of the body, eg type I
collagen (accounting for 90% of body collagen) found in bone, skin, tendon, ligaments,
cornea, internal organs or type II collagen found in cartilage, invertebral disc, notochord,
15 vitreous humour of the eye.
Proteins found in blood, including:
Plasma proteins such as fibrin, a~2 macro globulin, serum albumin, fibrinogen A,
20 fibrinogen B, serum amyloid protein A, heptaglobin, profilin, ubiquitin, uteroglobulin and
/^-microglobulin;
Enzymes and inhibitors such as plasminogen, lysozyme, cystatin C, alpha- 1 -antitrypsin
and pancreatic trypsin inhibitor. Plasminogen is the inactive precursor of the trypsin-like
25 serine protease plasmin. It is normally found circulating through the blood stream. When
plasminogen becomes activated and is converted to plasmin, it unfolds a potent enzymatic
domain that dissolves the fibrinogen fibers that entgangle the blood cells in a blood clot.
This is called fibrinolysis.
30 Immune system proteins, such as IgE, IgG, IgM.
Transport proteins such as retinol binding protein, a-l microglobulin.
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Defensins such as beta-defensin 1, Neutrophil defensins 1,2 and 3.
Proteins found at the blood brain barrier or in neural tissues, such as melanocortin
receptor, myelin, ascorbate transporter.
5
Transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins (see
US5977307);
brain capillary endothelial cell receptor, transferrin, transferrin receptor, insulin, insulin-
10 like growth factor 1 (IGF 1) receptor, insulin-like growth factor 2 (IGF 2) receptor,
insulin receptor.
Proteins localised to the kidney, such as polycystin, type IV collagen, organic anion
transporter Kl, Heymann's antigen.
15
Proteins localised to the liver, for example alcohol dehydrogenase, G250.
Blood coagulation factor X
cd antitrypsin
20 HNF la
Proteins localised to the lung, such as secretory component (binds IgA).
Proteins localised to the Heart, for example HSP 27. This is associated with dilated
25 cardiomyopathy.
Proteins localised to the skin, for example keratin.
Bone specific proteins, such as bone morphogenic proteins (BMPs), which are a subset of
30 the transforming growth factor |8 superfamily that demonstrate osteogenic activity.
Examples include BMP-2, -4, -5, -6, -7 (also referred to as osteogenic protein (OP-1) and
-8 (OP-2).
WO 2004/003019 PCT/GB2003/002804
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Tumour specific proteins, including human trophoblast antigen, herceptin receptor,
oestrogen receptor, cathepsins eg cathepsin B (found in liver and spleen).
Disease-specific proteins, such as antigens expressed only on activated T-cells: including
5 LAG-3 (lymphocyte activation gene), osteoprotegerin ligand (OPGL) see Nature 402,
304-309; 1999, OX40 (a member of the TNF receptor family, expressed on activated T
cells and the only costimulatory T cell molecule known to be specifically up-regulated in
human T cell leukaemia virus type-I (HTLV-I) -producing cells.) See J Immunol 2000
Jul 1;1 65(1): 263-70; Metalloproteases (associated with arthritis/cancers), including
10 CG65 12 Drosophila, human paraplegin, human FtsH, human AFG3L2, murine ftsH;
angiogenic growth factors, including acidic fibroblast growth factor (FGF-1), basic
fibroblast growth factor (FGF-2), Vascular endothelial growth factor / vascular
permeability factor (VEGF/VPF), transforming growth factor-a (TGF a), tumor necrosis
factor-alpha (TNF-ce), angiogenic interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-
15 derived endothelial growth factor (PD-ECGF), placental growth factor (P1GF), midkine
platelet-derived growth factor-BB (PDGF), fractalkine.
Stress proteins (heat shock proteins)
HSPs are normally found intracellularly. When they are found extracellularly, it is an
20 indicator that a cell has died and spilled out its contents. This unprogrammed cell death
(necrosis) only occurs when as a result of trauma, disease or injury and therefore in vivo,
extracellular HSPs trigger a response from the immune system that will fight infection
and disease. A dual specific which binds to extracellular HSP can be localised to a
disease site.
25
Proteins involved in Fc transport
Brambell receptor (also known as FcRB)
This Fc receptor has two functions, both of which are potentially useful for delivery
The functions are
30 (1) The transport of IgG from mother to child across the placenta
(2) the protection of IgG from degradation thereby prolonging its serum half life of
IgG. It is thought that the receptor recycles IgG from endosome.
WO 2004/003019 PCT/GB2003/002804
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See Holliger et ah Nat Biotechnol 1997 Jul;15(7):632-6.
Ligands according to the invention may designed to be specific for the above targets
without requiring any increase in or increasing half life in vivo. For example, ligands
according to the invention can be specific for targets selected from the foregoing which
are tissue-specific, thereby enabling tissue-specific targeting of the dual specific ligand,
or a dAb monomer that binds a tissue-specific therapeutically relevant target, irrespective
of any increase in half-life, although this may result. Moreover, where the ligand or dAb
monomer targets kidney or liver, this may redirect the ligand or dAb monomer to an
alternative clearance pathway in vivo (for example, the ligand may be directed away from
liver clearance to kidney clearance).
H: Use of multispecific ligands according to the second configuration of the
invention
Multispecific ligands according to the method of the second configuration of the present
invention may be employed in in vivo therapeutic and prophylactic applications, in vitro
and in vivo diagnostic applications, in vitro assay and reagent applications, and the like.
For example antibody molecules may be used in antibody based assay techniques, such as
ELIS A techniques, according to methods known to those skilled in the art.
As alluded to above, the multispecific ligands according to the invention are of use in
diagnostic, prophylactic and therapeutic procedures. Multispecific antibodies according to
the invention are of use diagnostically in Western analysis and in situ protein detection by
standard immunohistochemical procedures; for use in these applications, the ligands may
be labelled in accordance with techniques known to the art. In addition, such antibody
polypeptides may be used preparatively in affinity chromatography procedures, when
complexed to a chromatographic support, such as a resin. All such techniques are well
known to one of skill in the art.
Diagnostic uses of the closed conformation multispecific ligands according to the
invention include homogenous assays for analytes which exploit the ability of closed
conformation multispecific ligands to bind two targets in competition, such that two
WO 2004/003019 PCT/GB2003/002804
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targets cannot bind simultaneously (a closed conformation), or alternatively their ability
to bind two targets simultaneously (an open conformation).
A true homogenous immunoassay format has been avidly sought by manufacturers of
5 diagnostics and research assay systems used in drug discovery and development. The
main diagnostics markets include human testing in hospitals, doctor's offices and clinics,
commercial reference laboratories, blood banks, and the home, non-human diagnostics
(for example food testing, water testing, environmental testing, bio-defence, and
veterinary testing), and finally research (including drug development; basic research and
1 0 academic research) .
At present all these markets utilise immunoassay systems that are built around
chemiluminescent, ELISA, fluorescence or in rare cases radioimmunoassay
technologies. Each of these assay formats requires a separation step (separating bound
15 from un-bound reagents). In some cases, several separation steps are required. Adding
these additional steps adds reagents and automation, takes time, and affects the ultimate
outcome of the assays. In human diagnostics, the separation step may be automated,
which masks the problem, but does not remove it. The robotics, additional reagents,
additional incubation times, and the like add considerable cost and complexity. In drug
20 development, such as high throughput screening, where literally millions of samples are
tested at once, with very low levels of test molecule, adding additional separation steps
can eliminate the ability to perform a screen. However, avoiding the separation creates
too much noise in the read out. Thus, there is a need for a true homogenous format that
provides sensitivities at the range obtainable from present assay formats. Advantageously,
25 an assay possesses fully quantitative read-outs with high sensitivity and a large dynamic
range. Sensitivity is an important requirement, as is reducing the amount of sample
required. Both of these features are features that a homogenous system offers. This is
very important in point of care testing, and in drug development where samples are
precious. Heterogenous systems, as currently available in the art, require large quantities
30 of sample and expensive reagents
Applications for homogenous assays include cancer testing, where the biggest assay is
that for Prostate Specific Antigen, used in screening men for prostate cancer. Other
WO 2004/003019 PCT/GB2003/002804
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applications include fertility testing, which provides a series of tests for women
attempting to conceive including beta-hcg for pregnancy. Tests for infectious diseases,
including hepatitis, HIV, rubella, and other viruses and microorganisms and sexually
transmitted diseases. Tests are used by blood banks, especially tests for HIV, hepatitis A,
5 B, C, non A non B. Therapeutic drug monitoring tests include monitoring levels of
prescribed drugs in patients for efficacy and to avoid toxicity, for example digoxin for
arrhythmia, and phenobarbital levels in psychotic cases; theophylline for asthma.
Diagnostic tests are moreover useful in abused drug testing, such as testing for cocaine,
marijuana and the like. Metabolic tests are used for measuring thyroid function, anaemia
10 and other physiological disorders and functions.
The homogenous immunoassay format is moreover useful in the manufacture of standard
clinical chemistry assays. The inclusion of immunoassays and chemistry assays on the
same instrument is highly advantageous in diagnostic testing. Suitable chemical assays
15 include tests for glucose, cholesterol, potassium, and the like.
A further major application for homogenous immunoassays is drug discovery and
development: high throughput screening includes testing combinatorial chemistry
libraries versus targets in ultra high volume. Signal is detected, and positive groups then
20 split into smaller groups, and eventually tested in cells and then animals. Homogenous
assays may be used in all these types of test. In drug development, especially animal
studies and clinical trials heavy use of immunoassays is made. Homogenous assays
greatly accelerate and simplify these procedures. Other Applications include food and
beverage testing: testing meat and other foods for E. coli, salmonella, etc; water testing,
25 including testing at water plants for all types of contaminants including E. coli; and
veterinary testing.
In a broad embodiment, the invention provides a binding assay comprising a detectable
agent which is bound to a closed conformation multispecific ligand according to the
30 invention, and whose detectable properties are altered by the binding of an analyte to said
closed conformation multispecific ligand. Such an assay may be configured in several
different ways, each exploiting the above properties of closed conformation multispecific
ligands.
WO 2004/003019
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The assay relies on the direct or indirect displacement of an agent by the analyte, resulting
in a change in the detectable properties of the agent. For example, where the agent is an
enzyme which is capable of catalysing a reaction which has a detectable end-point, said
5 enzyme can be bound by the ligand such as to obstruct its active site, thereby inactivating
the enzyme. The analyte, which is also bound by the closed conformation multispecific
ligand, displaces the enzyme, rendering it active through freeing of the active site. The
enzyme is then able to react with a substrate, to give rise to a detectable event. In an
alternative embodiment, the ligand may bind the enzyme outside of the active site,
10 influencing the conformation of the enzyme and thus altering its activity. For example,
the structure of the active site may be constrained by the binding of the ligand, or the
binding of cofactors necessary for activity may be prevented.
The physical implementation of the assay may take any form known in the art. For
15 example, the closed conformation multispecific ligand/enzyme complex may be provided
on a test strip; the substrate may be provided in a different region of the test strip, and a
solvent containing the analyte allowed to migrate through the ligand/enzyme complex,
displacing the enzyme, and carrying it to the substrate region to produce a signal.
Alternatively, the ligand/enzyme complex may be provided on a test stick or other solid
20 phase, and dipped into an analyte/substrate solution, releasing enzyme into the solution in
response to the presence of analyte.
Since each molecule of analyte potentially releases one enzyme molecule, the assay is
quantitative, with the strength of the signal generated in a given time being dependent on
25 the concentration of analyte in the solution.
Further configurations using the analyte in a closed conformation are possible. For
example, the closed conformation multispecific ligand may be configured to bind an
enzyme in an allosteric site, thereby activating the enzyme. In such an embodiment, the
30 enzyme is active in the absence of analyte. Addition of the analyte displaces the enzyme
and removes allosteric activation, thus inactivating the enzyme.
WO 2004/003019 PCT/GB2003/002804
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In the context of the above embodiments which employ enzyme activity as a measure of
the analyte concentration, activation or inactivation of the enzyme refers to an increase or
decrease in the activity of the enzyme, measured as the ability of the enzyme to catalyse a
signal-generating reaction. For example, the enzyme may catalyse the conversion of an
undetectable substrate to a detectable form thereof. For example, horseradish peroxidase
is widely used in the art together with chromogenic or chemiluminescent substrates,
which are available commercially. The level of increase or decrease of the activity of the
enzyme may between 10% and 100%, such as 20%, 30%, 40%, 50%, 60%, 70%, 80% or
90%; in the case of an increase in activity, the increase may be more than 100%, i.e.
200%, 300%, 500% or more, or may not be measurable as a percentage if the baseline
activity of the inhibited enzyme is undetectable.
In a further configuration, the closed conformation multispecific ligand may bind the
substrate of an enzyme/substrate pair, rather than the enzyme. The substrate is therefore
unavailable to the enzyme until released from the closed conformation multispecific
ligand through binding of the analyte. The implementations for this configuration are as
for the configurations which bind enzyme.
Moreover, the assay may be configured to bind a fluorescent molecule, such as a
fluorescein or another fluorophore, in a conformation such that the fluorescence is
quenched on binding to the ligand. In this case, binding of the analyte to the ligand will
displace the fluorescent molecule, thus producing a signal. Alternatives to fluorescent
molecules which are useful in the present invention include luminescent agents, such as
luciferin/luciferase, and chromogenic agents, including agents commonly used in
immunoassays such as BDRP.
Therapeutic and prophylactic uses of multispecific ligands prepared according to the
invention involve the administration of ligands according to the invention to a recipient
mammal, such as a human. Multi-specificity can allow antibodies to bind to multimeric
antigen with great avidity. Multispecific ligands can allow thecross- linking of two
antigens, for example in recruiting cytotoxic T-cells to mediate the killing of tumour cell
lines.
WO 2004/003019 PCT/GB2003/002804
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Substantially pure ligands or binding proteins thereof, for example dAb monomers, of at
least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to
99% or more homogeneity is most preferred for pharmaceutical uses, especially when the
mammal is a human. Once purified, partially or to homogeneity as desired, the ligands
5 may be used diagnostically or therapeutically (including extracorporeally) or in
developing and performing assay procedures, immunofluorescent stainings and the like
(Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II,
Academic Press, NY).
10 The ligands or binding proteins thereof, for example dAb monomers, of the present
invention will typically find use in preventing, suppressing or treating inflammatory
states, allergic hypersensitivity, cancer, bacterial or viral infection, and autoimmune
disorders (which include, but are not limited to, Type I diabetes, asthma, multiple
sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and
15 myasthenia gravis).
In the instant application, the term "prevention" involves administration of the protective
composition prior to the induction of the disease. "Suppression" refers to administration
of the composition after an inductive event, but prior to the clinical appearance of the
20 disease. "Treatment" involves administration of the protective composition after disease
symptoms become manifest.
Animal model systems which can be used to screen the effectiveness of the antibodies or
binding proteins thereof in protecting against or treating the disease are available.
25 Methods for the testing of systemic lupus erythematosus (SLE) in susceptible mice are
known in the art (Knight et al. (1978) J. Exp. Med., 147: 1653; Reinersten et al. (1978)
New Eng. J. Med., 299: 515). Myasthenia Gravis (MG) is tested in SJL/J female mice by
inducing the disease with soluble AchR protein from another species (Lindstrom et al.
(1988) Adv. Immunol., 42: 233). Arthritis is induced in a susceptible strain of mice by
30 injection of Type II collagen (Stuart et al. (1984) Ann. Rev. Immunol., 42: 233). A model
by which adjuvant arthritis is induced in susceptible rats by injection of mycobacterial
heat shock protein has been described (Van Eden et al. (1988) Nature, 331: 171).
Thyroiditis is induced in mice by administration of thyroglobulin as described (Maron et
WO 2004/003019 PCT/GB2003/002804
78
al (1980) J. Exp. Med., 152: 1115). Insulin dependent diabetes mellitus (IDDM) occurs
naturally or can be induced in certain strains of mice such as those described by
Kanasawa et al (1984) Diabetologia, 27: 113. EAE in mouse and rat serves as a model
for MS in human. In this model, the demyelinating disease is induced by administration
5 of myelin basic protein (see Paterson (1986) Textbook of Immunopathology, Mischer et
al, eds., Grune and Stratton, New York, pp. 179-213; McFarlin et al (1973) Science,
179: 478: and Satoh et al (1987) J. Immunol, 138: 179).
Generally, the present ligands will be utilised in purified form together with
10 pharmacologically appropriate carriers. Typically, these carriers include aqueous or
alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or
buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable
adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from
15 thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.
Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers,
such as those based on Ringer's dextrose. Preservatives and other additives, such as
antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack
20 (1 982) Remington 's Pharmaceutical Sciences, 1 6th Edition).
The ligands of the present invention may be used as separately administered compositions
or in conjunction with other agents. These can include various immunotherapeutic drugs,
such as cylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins.
25 Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents
in conjunction with the ligands of the present invention, or even combinations of lignds
according to the present invention having different specificities, such as ligands selected
using different target antigens or epitopes, whether or not they are pooled prior to
administration.
30
The route of administration of pharmaceutical compositions according to the invention
may be any of those commonly known to those of ordinary skill in the art. For therapy,
including without limitation immunotherapy, the selected ligands thereof of the invention
WO 2004/003019 PCT/GB2003/002804
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can be administered to any patient in accordance with standard techniques. The
administration can be by any appropriate mode, including parenterally, intravenously,
intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also,
appropriately, by direct infusion with a catheter. The dosage and frequency of
5 administration will depend on the age, sex and condition of the patient, concurrent
administration of other drugs, counterindications and other parameters to be taken into
account by the clinician.
The ligands of this invention can be lyophilised for storage and reconstituted in a suitable
10 carrier prior to use. This technique has been shown to be effective with conventional
immunoglobulins and art-known lyophilisation and reconstitution techniques can be
employed. It will be appreciated by those skilled in the art that lyophilisation and
reconstitution can lead to varying degrees of antibody activity loss (e.g. with conventional
immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies)
15 and that use levels may have to be adjusted upward to compensate.
The compositions containing the present ligands or a cocktail thereof can be administered
for prophylactic and/or therapeutic treatments. In certain therapeutic applications, an
adequate amount to accomplish at least partial inhibition, suppression, modulation,
20 killing, or some other measurable parameter, of a population of selected cells is defined as
a "therapeutically-effective dose". Amounts needed to achieve this dosage will depend
upon the severity of the disease and the general state of the patient's own immune system,
but generally range from 0.005 to 5.0 mg of ligand, e.g. antibody, receptor (e.g. a T-cell
receptor) or binding protein thereof per kilogram of body weight, with doses of 0.05 to
25 2.0 mg/kg/dose being more commonly used. For prophylactic applications, compositions
containing the present ligands or cocktails thereof may also be administered in similar or
slightly lower dosages.
Treatment performed using the compositions described herein is considered "effective" if
30 one or more symptoms is reduced (e.g., by at least 10% or at least one point on a clinical
assessment scale), relative to such symptoms present before treatment, or relative to such
symptoms in an individual (human or model animal) not treated with such composition.
Symptoms will obviously vary depending upon the disease or disorder targeted, but can
WO 2004/003019 PCT/GB2003/002804
80
be measured by an ordinarily skilled clinician or technician. Such symptoms can be
measured, for example, by monitoring the level of one or more biochemical indicators of
the disease or disorder (e.g., levels of an enzyme or metabolite correlated with the
disease, ' affected cell numbers, etc.), by monitoring physical manifestations (e.g.,
5 inflammation, tumor size, etc.), or by an accepted clinical assessment scale, for example,
the Expanded Disability Status Scale (for multiple sclerosis), the Irvine Inflammatory
Bowel Disease Questionnaire (32 point assessment evaluates quality of life with respect
to bowel function, systemic symptoms, social function and emotional status - score ranges
from 32 to 224, with higher scores indicating a better quality of life), the Quality of Life
10 Rheumatoid Arthritis Scale, or other accepted clinical assessment scale as known in the
field. A sustained (e.g., one day or more, preferably longer) reduction in disease or
disorder symptoms by at least 1 0% or by one or more points on a given clinical scale is
indicative of "effective" treatment. Similarly, prophylaxis performed using a composition
as described herein is "effective" if the onset or severity of one or more symptoms is
1 5 delayed, reduced or abolished relative to such symptoms in a similar individual (human or
animal model) not treated with the composition.
A composition containing a ligand or cocktail thereof according to the present invention
may be utilised in prophylactic and therapeutic settings to aid in the alteration,
20 inactivation, killing or removal of a select target cell population in a mammal. In addition,
the selected repertoires of polypeptides described herein may be used extracorporeally or
in vitro selectively to kill, deplete or otherwise effectively remove a target cell population
from a heterogeneous collection of cells. Blood from a mammal may be combined
extracorporeally with the ligands, e.g. antibodies, cell-surface receptors or binding
25 proteins thereof whereby the undesired cells are killed or otherwise removed from the
blood for return to the mammal in accordance with standard techniques.
I: Use of half-life enhanced dual-specific ligands according to the invention
30
Dual-specific ligands according to the method of the present invention may be employed
in in vivo therapeutic and prophylactic applications, in vivo diagnostic applications and
the like.
WO 2004/003019
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Therapeutic and prophylactic uses of dual-specific ligands prepared according to the
invention involve the administration of ligands according to the invention to a recipient
mammal, such as a human. Dual specific antibodies according to the invention comprise
5 at least one specificity for a half-life enhancing molecule; one or more further specificities
may be directed against target molecules. For example, a dual-specific IgG may be
specific for four epitopes, one of which is on a half-life enhancing molecule. Dual-
specificity can allow antibodies to bind to multimeric antigen with great avidity. Dual-
specific antibodies can allow the cross-linking of two antigens, for example in recruiting
10 cytotoxic T-cells to mediate the killing of tumour cell lines.
Substantially pure ligands or binding proteins thereof, such as dAb monomers, of at least
90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or
more homogeneity is most preferred for pharmaceutical uses, especially when the
15 mammal is a human. Once purified, partially or to homogeneity as desired, the ligands
may be used diagnostically or therapeutically (including extracorporeal^) or in
developing and performing assay procedures, immunofluorescent stainings and the like
(Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II,
Academic Press, NY).
20
The ligands of the present invention will typically find use in preventing, suppressing or
treating inflammatory states, allergic hypersensitivity, cancer, bacterial or viral infection,
and autoimmune disorders (which include, but are not limited to, Type I diabetes,
multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease
25 and myasthenia gravis).
In the instant application, the term "prevention" involves administration of the protective
composition prior to the induction of the disease. "Suppression" refers to administration
of the composition after an inductive event, but prior to the clinical appearance of the
30 disease. "Treatment" involves administration of the protective composition after disease
symptoms become manifest.
WO 2004/003019 PCT/GB2003/002804
82
Animal model systems which can be used to screen the effectiveness of the dual specific
ligands in protecting against or treating the disease are available. Methods for the testing
of systemic lupus erythematosus (SLE) in susceptible mice are known in the art (Knight
et al (1978) J. Exp. Med., 147: 1653; Reinersten et al. (1978) New Eng. J. Med., 299:
5 515). Myasthenia Gravis (MG) is tested in SJL/J female mice by inducing the disease
with soluble AchR protein from another species (Lindstrom et al. (1988) Adv. Immunol.,
42: 233). Arthritis is induced in a susceptible strain of mice by injection of Type II
collagen (Stuart et al. (1984) Ann. Rev. Immunol., 42: 233). A model by which adjuvant
arthritis is induced in susceptible rats by injection of mycobacterial heat shock protein has
10 been described (Van Eden et al. (1988) Nature, 331: 171). Thyroiditis is induced in mice
by administration of thyroglobulin as described (Maron et al. (1980) J. Exp. Med., 152:
1115). Insulin dependent diabetes mellitus (IDDM) occurs naturally or can be induced in
certain strains of mice such as those described by Kanasawa et al. (1984) Diabetologia,
27: 113. EAE in mouse and rat serves as a model for MS in human. In this model, the
15 demyelinating disease is induced by administration of myelin basic protein (see Paterson
(1986) Textbook of Immunopathology, Mischer et al., eds., Grune and Stratton, New
York, pp. 179-213; McFarlin et al. (1973) Science, 179: 478: and Satoh et al. (1987) J.
Immunol., 138: 179).
20 Dual specific ligands according to the invention and dAb monomers able to bind to
extracellular targets involved in endocytosis (e.g. Clathrin) enable dual specific ligands to
be endocytosed, enabling another specificity able to bind to an intracellular target to be
delivered to an intracellular environment. This strategy requires a dual specific ligand
with physical properties that enable it to remain functional inside the cell. Alternatively,
25 if the final destination intracellular compartment is oxidising, a well folding ligand may
not need to be disulphide free.
Generally, the present dual specific ligands will be utilised in purified form together with
pharmacologically appropriate carriers. Typically, these carriers include aqueous or
30 alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or
buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable
WO 2004/003019 PCT/GB2003/002804
83
adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen
from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and
alginates.
5 Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishes,
such as those based on Ringer's dextrose. Preservatives and other additives, such as
4
antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack
(1982) Remington's Pharmaceutical Sciences, 16th Edition).
10 The ligands of the present invention may be used as separately administered compositions
or in conjunction with other agents. These can include various immunotherapeutic drugs,
such as cylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins.
Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents
in conjunction with the ligands of the present invention.
15
The route of administration of pharmaceutical compositions according to the invention
may be any of those commonly known to those of ordinary skill in the art. For therapy,
including without limitation immunotherapy, the ligands of the invention can be
administered to any patient in accordance with standard techniques. The administration
20 can be by any appropriate mode, including parenterally, intravenously, intramuscularly,
intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct
infusion with a catheter. The dosage and frequency of administration will depend on the
age, sex and condition of the patient, concurrent administration of other drugs,
counterindications and other parameters to be taken into account by the clinician.
25
The ligands of the invention can be lyophilised for storage and reconstituted in a suitable
carrier prior to use. This technique has been shown to be effective with conventional
immunoglobulins and art-known lyophilisation and reconstitution techniques can be
employed. It will be appreciated by those skilled in the art that lyophilisation and
30 reconstitution can lead to varying degrees of antibody activity loss (e.g. with conventional
immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies)
and that use levels may have to be adjusted upward to compensate.
WO 2004/003019 PCT/GB2003/002804
84
The compositions containing the present ligands or a cocktail thereof can be
administered for prophylactic and/or therapeutic treatments. In certain therapeutic
applications, an adequate amount to accomplish at least partial inhibition, suppression,
modulation, killing, or some other measurable parameter, of a population of selected cells
5 is defined as a "therapeutically-effective dose". Amounts needed to achieve this dosage
will depend upon the severity of the disease and the general state of the patient's own
immune system, but generally range from 0.005 to 5.0 mg of ligand per kilogram of body
weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. For
prophylactic applications, compositions containing the present ligands or cocktails thereof
10 may also be administered in similar or slightly lower dosages.
A composition containing a ligand according to the present invention may be utilised in
prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or
removal of a select target cell population in a mammal.
15
In addition, the selected repertoires of polypeptides described herein may be used
extracorporeal^ or in vitro selectively to kill, deplete or otherwise effectively remove a
target cell population from a heterogeneous collection of cells. Blood from a mammal
may be combined extracorporeally with the ligands, e.g. antibodies, cell- surface
20 receptors or binding proteins thereof whereby the undesired cells are killed or otherwise
removed from the blood for return to the mammal in accordance with standard
techniques.
The invention is further described, for the purposes of illustration only, in the following
25 examples. As used herein, for the purposes of dAb nomenclature, human TNFa is
referred to as TAR1 and human TNFa receptor 1 (p55 receptor) is referred to as TAR2.
Example 1. Selection of a dual specific scFv antibody (K8) directed against human
30 serum albumin (HSA) and P-galactosidase -gal)
This example explains a method for making a dual specific antibody directed against 0-
gal and HSA in which a repertoire of V K variable domains linked to a germline (dummy)
WO 2004/003019 PCT/GB2003/002804
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Vh domain is selected for binding to p-gal and a repertoire of Vh variable domains
linked to a germline (dummy) V K domain is selected for binding to HSA. The selected
variable Vh HSA and V K p-gal domains are then combined and the antibodies selected for
binding to P-gal and HSA. HSA is a half-life increasing protein found in human blood.
Four human phage antibody libraries were used in this experiment.
Library 1 Germline V K /DVT V H 8.46 x 10 7
Library 2 Germline V K /NNK V H 9.64 x 10 7
Library 3 Germline V H /DVT V K 1 .47 x 1 0 8
Library 4 Germline V H /NNK V K 1.45x1 0 8
All libraries are based on a single human framework for Vh (V3-23/DP47 and J H 4b) and
V K (012/02/DPK9 and J K 1) with side chain diversity incorporated in complementarity
determining regions (CDR2 and CDR3).
Library 1 and Library 2 contain a dummy V K sequence, whereas the sequence of Vh is
diversified at positions H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97 and H98
(DVT or NNK encoded, respectively) (Figure 1). Library 3 and Library 4 contain a
dummy Vh sequence, whereas the sequence of V K is diversified at positions L50, L53,
L91, L92, L93, L94 and L96 (DVT or NNK encoded, respectively) (Figure 1). The
libraries are in phagemid pIT2/ScFv format (Figure 2) and have been preselected for
binding to generic ligands, Protein A and Protein L, so that the majority of clones in the
unselected libraries are functional. The sizes of the libraries shown above correspond to
the sizes after preselection. Library 1 and Library 2 were mixed prior to selections on
antigen to yield a single Vn/duinmy V K library and Library 3 and Library 4 were mixed
to form a single V K /dummy Vh library.
Three rounds of selections were performed on P-gal using V K /dummy V H library and
three rounds of selections were performed on HSA using Vn/diunmy V K library. In the
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case of p-gal the phage titres went up from 1.1 x 10^ in the first round to 2.0 x 10^ in the
third round. In the case of HSA the phage titres went up from 2 x l O^ in the first round to
1.4 x 1()9 in the third round. The selections were performed as described by Griffith et al. 9
(1993), except that KM13 helper phage (which contains a pill protein with a protease
5 cleavage site between the D2 and D3 domains) was used and phage were eluted with 1
mg/ml trypsin in PBS. The addition of trypsin cleaves the pill proteins derived from the
helper phage (but not those from the phagemid) and elutes bound scFv-phage fusions by
cleavage in the c-myc tag (Figure 2), thereby providing a further enrichment for phages
expressing functional scFvs and a corresponding reduction in background (Kristensen &
10 Winter, Folding & Design 3: 321-328, Jul 9, 1998). Selections were performed using
immunotubes coated with either HSA or P-gal at lOOjag/ml concentration.
To check for binding, 24 colonies from the third round of each selection were screened by
monoclonal phage ELISA. Phage particles were produced as described by Harrison et aL,
15 Methods Enzymol. 1996;267:83-109. 96-well ELISA plates were coated with 100pl of
HSA or P-gal at 10|ag/ml concentration in PBS overnight at 4°C. A standard ELISA
protocol was followed (Hoogenboom et aL, 1991) using detection of bound phage with
anti-M13-HRP conjugate. A selection of clones gave ELISA signals of greater than 1.0
with 50|ixl supernatant.
20
Next, DNA preps were made from V H /dummy V K library selected on HSA and from
V K /dummy Vh library selected on p-gal using the QIAprep Spin Miniprep kit (Qiagen).
To access most of the diversity, DNA preps were made from each of the three rounds of
selections and then pulled together for each of the antigens. DNA preps were then
25 digested with SalZ/Not/ overnight at 37°C. Following gel purification of the fragments,
V K chains from the V K /dummy Vh library selected on p-gal were ligated in place of a
dummy V K chain of the VH/dummy V K library selected on HSA creating a library of 3.3
x 10 9 clones.
30 This library was then either selected on HSA (first round) and P-gal (second round),
HSA/p-gal selection, or on P-gal (first round) and HSA (second round), p-gal/HSA
WO 2004/003019 PCT/GB2003/002804
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selection. Selections were performed as described above. Li each case after the second
round 48 clones were tested for binding to HSA and P-gal by the monoclonal phage
ELISA (as described above) and by ELISA of the soluble scFv fragments. Soluble
antibody fragments were produced as described by Harrison et ah, (1996), and standard
5 ELISA protocol was followed Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133,
except that 2% Tween/PBS was used as a blocking buffer and bound scFvs were detected
with Protein L-HRP. Three clones (E4, E5 and E8) from the HSA/ p-gal selection and two
clones (K8 and K10) from the p-gal/HSA selection were able to bind both antigens. scFvs
from these clones were PCR amplified and sequenced as described by Ignatovich et al.,
10 (1999) J Mol Biol 1999 Nov 26;294(2):457-65, using the primers LMB3 and pHENseq.
Sequence analysis revealed that all clones were identical. Therefore, only one clone
encoding a dual specific antibody (K8) was chosen for further work (Figure 3).
15 Example 2. Characterisation of the binding properties of the K8 antibody.
Firstly, the binding properties of the K8 antibody were characterised by the monoclonal
phage ELISA. A 9 6 -well plate was coated with 100pl of HSA and P-gal alongside with
alkaline phosphatase (APS), bovine serum albumin (BSA), peanut agglutinin, lysozyme
20 and cytochrome c (to check for cross-reactivity) at 10jag/ml concentration in PBS
overnight at 4°C. The phagemid from K8 clone was rescued with KM13 as described by
Harrison et aL 9 (1996) and the supernatant (50jli1) containing phage assayed directly. A
standard ELISA protocol was followed (Hoogenboom et ah, 1991) using detection of
bound phage with anti-M13-HRP conjugate. The dual specific K8 antibody was found to
25 bind to HSA and P-gal when displayed on the surface of the phage with absorbance
signals greater than 1.0 (Figure 4). Strong binding to BSA was also observed (Figure 4).
Since HSA and BSA are 76% homologous on the amino acid level, it is not surprising
that K8 antibody recognised both of these structurally related proteins. No cross-reactivity
with other proteins was detected (Figure 4).
30
Secondly, the binding properties of the K8 antibody were tested in a soluble scFv ELISA.
Production of the soluble scFv fragment was induced by IPTG as described by Harrison
et al., (1996). To determine the expression levels of K8 scFv, the soluble antibody
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fragments were purified from the supernatant of 50ml inductions using Protein A-
Sepharose columns as described by Harlow and Lane, Antibodies: a Laboratory Manual,
(1988) Cold Spring Harbor. OD28O was ^en measured and the protein concentration
calculated as described by Sambrook et al. 9 (1989). K8 scFv was produced in supernatant
5 at 19mg/L
A soluble scFv ELISA was then performed using known concentrations of the K8
antibody fragment. A 9 6- well plate was coated with lOOjal of HSA, BSA and p-gal at
lOjag/ml and lOOpl of Protein A at l|ag/ml concentration. 50|al of the serial dilutions of
10 the K8 scFv was applied and the bound antibody fragments were detected with Protein L-
HRP. ELISA results confirmed the dual specific nature of the K8 antibody (Figure 5).
To confirm that binding to P-gal is determined by the V K domain and binding to
HSA/BSA by the V H domain of the K8 scFv antibody, the V K domain was cut out from
15 K8 scFv DNA by SalZ/Not/ digestion and ligated into a SalZ/Not/ digested pIT2 vector
containing dummy V H chain (Figures 1 and 2). Binding characteristics of the resulting
clone K8V K /dummy V H were analysed by soluble scFv ELISA. Production of the soluble
scFv fragments was induced by EPTG as described by Harrison et al. y (1996) and the
supernatant (50^i) containing scFvs assayed directly. Soluble scFv ELISA was performed
20 as described in Example 1 and the bound scFvs were detected with Protein L-HRP. The
ELISA results revealed that this clone was still able to bind P-gal, whereas binding to
BSA was abolished (Figure 6).
Example 3. Selection of single Vjj domain antibodies antigens A and B and single
25 V K domain antibodies directed against antigens C and D.
This example describes a method for making single Vjj domain antibodies directed
against antigens A and B and single V K domain antibodies directed against antigens C
and D by selecting repertoires of virgin single antibody variable domains for binding to
30 these antigens in the absence of the complementary variable domains.
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Selections and characterisation of the binding clones is performed as described
previously (see Example 5, PCT/GB 02/003014). Four clones are chosen for further
work:
5 VHl-AntiAV H
VH2-AntiB Vh
VK1 - Anti C V K
VK2~AntiD V K
10 The procedures described above in Examples 1-3 may be used, in a similar manner as that
described, to produce dimer molecules comprising combinations of V H domains (i.e., V H -
V H ligands) and cominations of Vl domains (V l -Vl ligands).
Example 4. Creation and characterisation of the dual specific ScFv antibodies
15 (VH1/VH2 directed against antigens A and B and VK1/VK2 directed against
antigens C and D).
This example demonstrates that dual specific ScFv antibodies (VH1/VH2 directed against
antigens A and B and VK1/VK2 directed against antigens C and D) could be created by
20 combining V K and V H single domains selected against respective antigens in a ScFv
vector.
To create dual specific antibody VH1/VH2, VH1 single domain is excised from variable
domain vector 1 (Figure 7) by NcollXhol digestion and ligated into NcoVXhol digested
25 variable domain vector 2 (Figure 7) to create VH1/ variable domain vector 2. VH2 single
domain is PGR amplified from variable domain vector 1 using primers to introduce Sail
restriction site to the 5' end and Notl restriction site to the 3 5 end. The PGR product is
then digested with Sail/ Notl and ligated into SaWNotl digested VH1/ variable domain
vector 2 to create VH1/VH2/ variable domain vector 2.
30
VK1 /VK2/ variable domain vector 2 is created in a similar way. The dual specific nature
of the produced VH1 /VH2 ScFv and VK1/VK2 ScFv is tested in a soluble ScFv ELISA
WO 2004/003019 PCT/GB2003/002804
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as described previously (see Example 6, PCT/GB 02/003014). Competition ELISA is
performed as described previously (see Example 8, PCT/GB 02/003014).
Possible outcomes:
5 -VH1/VH2 ScFv is able to bind antigens A and B simultaneously
-VK1/VK2 ScFv is able to bind antigens C and D simultaneously
-VH1/VH2 ScFv binding is competitive (when bound to antigen A, VH1/VH2 ScFv
cannot bind to antigen B)
-VK1/VK2 ScFv binding is competitive (when bound to antigen C, VK1/VK2 ScFv
10 cannot bind to antigen D)
Example 5. Construction of dual specific VH1/VH2 Fab and VK1/VK2 Fab and
analysis of their binding properties,
15 To create VH1/VH2 Fab, VH1 single domain is ligated into NcoVXfiol digested CH
vector (Figure 8) to create VH1/CH and VH2 single domain is ligated into SaWNoil
digested CK vector (Figure 9) to create VH2/CK. Plasmid DNA from VH1/CH and
VH2/CK is used to co-transform competent E. coli cells as described previously (see
Example 8, PCT/GB 02/003 01 4).
20
The clone containing VH1/CH and VH2/CK plasmids is then induced by IPTG to
produce soluble VH1/VH2 Fab as described previously (see Example 8, PCT/GB
02/003014).
VK1/VK2 Fab is produced in a similar way.
Binding properties of the produced Fabs are tested by competition ELISA as described
previously (see Example 8, PCT/GB 02/003014).
30
Possible outcomes:
-VH1/VH2 Fab is able to bind antigens A and B simultaneously
-VK1/VK2 Fab is able to bind antigens C and D simultaneously
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-VH1/VH2 Fab binding is competitive (when bound to antigen A 5 VH1/VH2 Fab cannot
bind to antigen B)
-VK1/VK2 Fab binding is competitive (when bound to antigen C, VK1/VK2 Fab cannot
bind to antigen D)
5
Example 6
Chelating dAb Dimers
10 Summary
VH and VK homo-dimers are created in a dAb-linker~dAb format using flexible
polypeptide linkers. Vectors were created in the dAb linker-dAb format containing
glycine-serine linkers of different lengths 3U:(Gly4Ser) 3 , 5U:(Gly4Ser)5 ? 7U:(Gly4Ser)7.
Dimer libraries were created using guiding dAbs upstream of the linker: TAR1-5 (VK) 5
15 TAR1-27(VK) 3 TAR2-5(VH) or TAR2-6(VK) and a library of corresponding second
dAbs after the linker. Using this method, novel dimeric dAbs were selected. The effect of
dimerisation on antigen binding was determined by ELISA and BIAcore studies and in
cell neutralisation and receptor binding assays. Dimerisation of both TAR1-5 and TAR1-
27 resulted in significant improvement in binding affinity and neutralisation levels.
20
1.0 Methods
1.1 Library generation
1.1.1 Vectors
pEDASU, pEDA5U and pEDA7U vectors were designed to introduce different linker
25 lengths compatible with the dAb - linker- dAb format. For pEDA3U, sense and anti-sense
73-base pair oligo linkers were annealed using a slow annealing program (95°C-5mins,
80°C-10mins, 70°C-15mins, 56°C-15mins ? 42°C until use) in buffer containing
O.lMNaCl, lOmM Tris-HCl pH7.4 and cloned using the Xltol and Notl restriction sites.
The linkers encompassed 3 (Gly4Ser) units and a stuffer region housed between Sail and
30 Notl cloning sites (scheme 1). In order to reduce the possibility of monomelic dAbs
being selected for by phage display, the stuffer region was designed to include 3 stop
codons, a Sacl restriction site and a frame shift mutation to put the region out of frame
when no second dAb was present. For pEDASU and 7U due to the length of the linkers
WO 2004/003019 PCT/GB2003/002804
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required, overlapping oligo -linkers were designed for each vector, annealed and
elongated using Klenow. The fragment was then purified and digested using the
appropriate enzymes before cloning using tho Xhol and Notl restriction sites.
Ncol
I
Xhol
Stuffer 1
Linker:
3U
5U
Sail
7U
1
Notl
Stuffer 2
Scheme 1
1.1.2 Library preparation
The N-terminal V gene corresponding to the guiding dAb was cloned upstream of the
10 linker using Ncol and Xhol restriction sites. VH genes have existing compatible sites,
however cloning VK genes required the introduction of suitable restriction sites. This was
achieved by using modifying PCR primers (VK-DLIBF: 5' cggccatggcgtcaacggacat;
VKXholR: 5' atgtgcgctcgagcgtttgattt 3') in 30 cycles of PCR amplification using a 2:1
mixture of SuperTaq (HTBiotechnology Ltd)and pfu turbo (Stratagene). This maintained
15 the Ncol site at the 5' end while destroying the adjacent Sail site and introduced the
Xhol site at the 3' end. 5 guiding dAbs were cloned into each of the 3 dimer vectors:
TAR1-5 (VK), TAR1-27(VK), TAR2-5(VH), TAR2-6(VK) and TAR2-7(VK). All
constructs were verified by sequence analysis.
20 Having cloned the guiding dAbs upstream of the linker in each of the vectors (pEDA3U,
5U and 7U): TAR1-5 (VK), TAR1-27(VK), TAR2-5(VH) or TAR2-6(VK) a library of
corresponding second dAbs were cloned after the linker. To achieve this, the
complimentary dAb libraries were PCR amplified from phage recovered from round 1
selections of either a VK library against Human TNFa (at approximately 1 x 10 6 diversity
25 after round 1) when TAR 1-5 or TAR1-27 are the guiding dAbs, or a VH or VK library
against human p55 TNF receptor (both at approximately 1 x 10 5 diversity after round 1)
when TAR2-5 or TAR2-6 respectively are the guiding dAbs. For VK libraries PCR
amplification was conducted using primers in 30 cycles of PCR amplification using a 2:1
mixture of SuperTaq and pfu turbo. VH libraries were PCR amplified using primers in
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order to introduce a Sail restriction site at the 5' end of the gene. The dAb library PCRs
were digested with the appropriate restriction enzymes, ligated into the corresponding
vectors down stream of the linker, using Sall/Notl restriction sites and electroporated
into freshly prepared competent TGI cells.
5
The titres achieved for each library are as follows:
TAR1-5: pEDA3U = 4xl0 8 , pEDASU = 8xl0 7 , pEDA7U = 1x10 s
TAR1-27: pEDASU = 6.2xl0 8 , pEDASU - 1x10 s , pEDA7U = lxlO 9
TAR2h-5 : pEDA3U = 4xl0 7 , pEDA5U - 2 x 10 8 , pEDA7U = 8xl0 7
10 TAR2h-6: pEDA3U = 7.4xl0 8 5 pEDASU = 1.2 x 10 8 , pEDA7U = 2.2xl0 8
1.2 Selections
1.2.1 TNFa
Selections were conducted using human TNFa passively coated on immunotubes.
15 Briefly, Immunotubes are coated overnight with l-4mls of the required antigen. The
immunotubes were then washed 3 times with PBS and blocked with 2%milk powder in
PBS for l~2hrs and washed a further 3 times with PBS. The phage solution is diluted in
2%milk powder in PBS and incubated at room temperature for 2hrs. The tubes are then
washed with PBS and the phage eluted with lmg/ml trypsin-PBS. Three selection
20 strategies were investigated for the TAR1-5 dimer libraries. The first round selections
were carried out in immunotubes using human TNFa coated at ljixg/ml or 20|Lig/ml with
20 washes in PBS 0.1%Tween. TGI cells are infected with the eluted phage and the titres
are determined (eg, Marks et al J Mol Biol. 1991 Dec 5;222(3):581-97, Richmann et al
Biochemistry. 1993 Aug 31;32(34):8848-55).
25
The titres recovered were:
pEDA3U = 2.8xl0 7 (ljig/ml TNF) 1.5xl0 8 (20|ag/mlTNF),
pEDASU = 1.8xl0 7 (l|ng/ml TNF), 1.6xl0 8 (20jag/ml TNF)
pEDA7U = 8xl0 6 (ljLig/ml TNF), 7xl0 7 (20jag/ml TNF).
30
The second round selections were carried out using 3 different methods.
1. In immunotubes, 20 washes with overnight incubation followed by a further 10
washes.
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2. In immunotubes, 20 washes followed by lhr incubation at RT in wash buffer
with (lp,g/ml TNFa) and 10 further washes.
3. Selection on streptavidin beads using 33 pmoles biotinylated human TNFa
(Henderikx et aL 9 2002, Selection of antibodies against biotinylated antigens.
Antibody Phage Display : Methods and protocols, Ed. O'Brien and Atkin, Humana
Press). Single clones from round 2 selections were picked into 96 well plates and
crude supernatant preps were made in 2ml 96 well plate format.
Round 1
Round 2
Round 2
Round 2
Human
selection
selection
selection
TNFaimmuno
method 1
method 2
method 3
tube coating
concentration
pEDA3U
l|_ig/ml
1 x 10 y
1.8 x 10 y
2.4 x 10 10
pEDA3U
20p,g/ml
6x 10 y
1.8 x 10 10
8.5 x 10 10
pEDA5U
l|j.g/ml
9x 10 a
1.4 x 10 y
2.8 x 10 10
pEDA5U
20u.g/ml
9.5 x 10 y
8.5 x 10 y
2.8 x 10 10
pEDA7U
l(j.g/ml
7.8 x 10*
1.6 x 10 8
4x 10 10
pEDA7U
20u.g/ml
1 x 10 10
8x 10 y
1.5 x 10 10
For TAR 1-27, selections were carried out as described previously with the following
modifications. The first round selections were carried out in immunotubes using human
TNFa coated at ljag/ml or 20|ug/ml with 20 washes in PBS 0.1%Tween. The second
round selections were carried out in immunotubes using 20 washes with overnight
incubation followed by a further 20 washes. Single clones from round 2 selections were
picked into 96 well plates and crude supernatant preps were made in 2ml 96 well plate
format.
TAR 1-2 7 titres are as follows
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PCT/GB2003/002804
Human
TNFaimmunotu.be
coating cone
Round 1
Round 2
pEDA3U
lug/ml
4x 10 y
6x 10 y
pEDA3U
20ug/ml
5x 10 y
4.4 x 10 1U
pEDA5U
lug/ml
1.5 x 10 9
1.9 x 10 1U
pEDA5U
20ug/ml
3.4 x 10 y
3.5 x 10 1U
pEDA7U
lug/ml
2.6 x 10 y
5 x 10 y
pEDA7U
20(j.g/ml
7x 10 y
1.4 x 10 1U
1.2.2 TNF RECEPTOR 1 (p55 RECEPTOR; TAR2)
Selections were conducted as described previously for the TAR2h-5 libraries only. 3
rounds of selections were carried out in immunotubes using either ljag/ml human p55
TNF receptor or 10jng/ml human p55 TNF receptor with 20 washes in PBS 0.1%Tween
with overnight incubation followed by a further 20 washes. Single clones from round 2
and 3 selections were picked into 96 well plates and crude supernatant preps were made
in 2ml 96 well plate format.
TAR2h-5 titres are as follows:
Round 1 human
p55TNF
receptor
immunotube
coating
concentration
Round 1
Round 2
Round 3
pEDA3U
lu,g/ml
2.4 x 10 6
1.2 x 10'
1.9 x 10 y
pEDA3U
lOug/ml
3.1 x 10 7
7 x 10 7
1 x 10 y
pEDA5U
lug/ml
2.5 x 10 6
1.1 x 10 7
5.7 x 10 s
pEDA5U
lOug/ml
3.7 x 10 7
2.3 x 10 s
2.9 x 10 y
pEDA7U
lug/ml
1.3 x 10 6
1.3 x 10 7
1.4 x 10 y
pEDA7U
10|j,g/ml
1.6 x 10 7
1.9 x 10 7
3 x 10 1U
1.3 Screening
Single clones from round 2 or 3 selections were picked from each of the 3U, 5U and 7U
libraries from the different selections methods, where appropriate. Clones were grown in
2xTY with lOOjag/ml ampicillin and 1% glucose overnight at 37°C. A 1/100 dilution of
this culture was inoculated into 2mls of 2xTY with 100|ng/ml ampicillin and 0.1%
glucose in 2ml, 96 well plate format and grown at 37°C shaking until OD600 was
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approximately 0.9. The culture was then induced with ImM IPTG overnight at 30°C.
The supernatants were clarified by centrifugation at 4000rpm for 15 rnins in a sorval plate
centrifuge. The supernatant preps the used for initial screening.
5 1.3.1 ELISA
Binding activity of dimeric recombinant proteins was compared to monomer by Protein
A/L ELISA or by antigen ELISA. Briefly, a 96 well plate is coated with antigen or
Protein A/L overnight at 4°C. The plate washed with 0.05% Tween-PBS, blocked for 2hrs
with 2% Tween-PBS. The sample is added to the plate incubated for 1 hr at room
10 temperature. The plate is washed and incubated with the secondary reagent for lhr at
room temperature. The plate is washed and developed with TMB substrate. Protein A/L-
HRP or India-HRP was used as a secondary reagent. For antigen ELISAs, the antigen
concentrations used were l|ug/ml in PBS for Human TNFa and human THF receptor 1.
Due to the presence of the guiding dAb in most cases dimers gave a positive ELISA
15 signal therefore off rate determination was examined by BIAcore.
1.3.2 BIAcore
BIAcore analysys was conducted for TAR1-5 and TAR2h-5 clones. For screening,
Human TNFawas coupled to a CMS chip at high density (approximately 10000 RUs).
20 50 jlxI of Human TNFoc(50 jag/ml) was coupled to the chip at 5jal/min in acetate buffer -
pH5.5. Regeneration of the chip following analysis using the standard methods is not
possible due to the instability of Human TNFa, therefore after each sample was analysed,
the chip was washed for 1 Omins with buffer.
For TAR1-5, clones supernatants from the round 2 selection were screened by BIAcore.
25 48 clones were screened from each of the 3U, 5U and 7U libraries obtained using the
following selection methods:
Rl: 1/xg/ml human TNFa immunotube, R2 1 /xg/ml human TNFa immunotube, overnight
wash.
Rl: 20/xg/ml human TNFa immunotube, R2 20/xg/ml human TNFa immunotube,
30 overnight wash.
Rl: 1/xg/ml human TNFa immunotube, R2 33 pmoles biotinylated human TNFa on
beads.
Rl: 20/xg/ml human TNFa immunotube, R2 33 pmoles biotinylated human TNFa beads.
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PCT/GB2003/002804
For screening, human p55 TNF receptor was coupled to a CMS chip at high density
(approximately 4000 RUs). 100 jlxI of human p55 TNF receptor (10 (ag/ml) was coupled
to the chip at 5jjl/min in acetate buffer - pH5.5. Standard regeneration conditions were
5 examined ( glycine pH2 or pH3) but in each case antigen was removed from the surface
of the chip therefore as with TNFa, therefore after each sample was analysed, the chip
was washed for 1 Omins with buffer.
For TAR2-5, clones supernatants from the round 2 selection were screened.
48 clones were screened from each of the 3U, 5U and 7U libraries, using the following
1 o selection methods :
Rl: 1/zg/ml human p55 TNF receptor immunotube, R2 1/xg/ml human p55 TNF receptor
immunotube, overnight wash.
Rl: 10/xg/ml human p55 TNF receptor immunotube, R2 1 0/xg/ml human p55 TNF
receptor immunotube, overnight wash.
15
1.3.3 Receptor and Cell Assays
The ability of the dimers to neutralise in the receptor assay was conducted as follows:
Receptor binding
20 Anti-TNF dAbs were tested for the ability to inhibit the binding of TNF to recombinant
TNF receptor 1 (p55). Briefly, Maxisorp plates were incubated overnight with 30mg/ml
anti-human Fc mouse monoclonal antibody (Zymed, San Francisco, USA). The wells
were washed with phosphate buffered saline (PBS) containing 0.05% Tween-20 and then
blocked with 1% BS A in PBS before being incubated with lOOng/ml TNF receptor 1 Fc
25 fusion protein (R&D Systems, Minneapolis, USA). Anti-TNF dAb was mixed with TNF
which was added to the washed wells at a final concentration of lOng/ml. TNF binding
was detected with 0.2mg/ml biotinylated anti-TNF antibody (HyCult biotechnology,
Uben, Netherlands) followed by 1 in 500 dilution of horse radish peroxidase labelled
streptavidin (Amersham Biosciences, UK) and then incubation with TMB substrate (KPL,
30 Gaithersburg, USA). The reaction was stopped by the addition of HC1 and the absorbance
was read at 450nm. Anti-TNF dAb activity lead to a decrease in TNF binding and
therefore a decrease in absorbance compared with the TNF only control.
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L929 Cytotoxicity Assay
Anti-TNF dAbs were also tested for the ability to neutralise the cytotoxic activity of TNF
on mouse L929 fibroblasts (Evans, T. (2000) Molecular Biotechnology 15, 243-248).
Briefly, L929 cells plated in microtitre plates were incubated overnight with anti-TNF
5 dAb, lOOpg/ml TNF and lmg/ml actinomycin D (Sigma, Poole, UK). Cell viability was
measured by reading absorbance at 490nm following an incubation with [3-(4,5-
dimethylthiazol-2-yl)-5-(3-carbboxym^
(Promega, Madison, USA). Anti-TNF dAb activity lead to a decrease in TNF cytotoxicity
and therefore an increase in absorbance compared with the TNF only control.
10
In the initial screen, supernatants prepared for BIAcore analysis, described above, were
also used in the receptor assay. Further analysis of selected dimers was also conducted in
the receptor and cell assays using purified proteins.
15 HeLa IL-8 assay
Anti-TNFRl or anti-TNF alpha dAbs were tested for the ability to neutralise the
induction of IL-8 secretion by TNF in HeLa cells (method adapted from that of Akeson,
L. et al (1996) Journal of Biological Chemistry 271, 30517-30523, describing the
induction of IL-8 by IL-1 in HUVEC; here we look at induction by human TNF alpha and
20 we use HeLa cells instead of the HUVEC cell line). Briefly, HeLa cells plated in
microtitre plates were incubated overnight with dAb and 300pg/ml TNF. Post incubation
the supernatant was aspirated off the cells and IL-8 concentration measured via a
sandwich ELISA (R&D Systems). Anti-TNFRl dAb activity lead to a decrease in IL-8
secretion into the supernatant compared with the TNF only control.
25
The L929 assay is used throughout the following experiments; however, the use of the
HeLa IL-8 assay is preferred to measure anti-TNF receptor 1 (p55) ligands; the presence
of mouse p55 in the L929 assay poses certain limitations in its use.
30 1.4 Sequence analysis
Dimers that proved to have interesting properties in the BIAcore and the receptor assay
screens were sequenced. Sequences are detailed in the sequence listing.
WO 2004/003019 PCT/GB2003/002804
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1.5 Formatting
1.5.1 TAR1-5-19 dimers
The TAR1-5 dimers that were shown to have good neutralisation properties were re-
formatted and analysed in the cell and receptor assays. The TAR1-5 guiding dab was
5 substituted with the affinity matured clone TAR1-5-19. To achieve this TAR1-5 was
cloned out of the individual dimer pair and substituted with TAR1-5-19 that had been
amplified by PCR. In addition, TAR1-5-19 homodimers were also constructed in the 3U,
5U and 7U vectors. The N terminal copy of the gene was amplified by PCR and cloned as
described above and the C-terminal gene fragment was cloned using existing Sail and
10 Notl restriction sites.
1.5.2 Mutagenesis
The amber stop codon present in dAb2, one of the C-terminal dAbs in the TAR1-5 dimer
pairs was mutated to a glutamine by site-directed mutagenesis.
15
1.5.3 Fabs
The dimers containing TAR1-5 or TAR 1-5- 19 were re-formatted into Fab expression
vectors. dAbs were cloned into expression vectors containing either the CK or CH genes
using Sfil and Notl restriction sites and verified by sequence analysis. The CK vector is
20 derived from a pUC based ampicillin resistant vector and the CH vector is derived from a
pACYC chloramphenicol resistant vector. For Fab expression the dAb-CH and dAb-CK
constructs were co-transformed into HB2151 cells and grown in 2xTY containing 0.1%
glucose, 100jj,g/ml ampicillin and 10/xg/ml chloramphenicol.
25 1.5.3 Hinge dimerisation
Dimerisation of dAbs via cystine bond formation was examined. A short sequence of
amino acids EPKSGDKTHTCPPCP a modified form of the human IgGCl hinge was
engineered at the C terminal region on the dAb. An oligo linker encoding for this
sequence was synthesised and annealed, as described previously. The linker was cloned
30 into the pEDA vector containing TAR1-5-19 using Xliol and Notl restriction sites.
Dimerisation occurs in situ in the periplasm.
1.6 Expression and purification
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1.6.1 Expression
Supernatants were prepared in the 2ml ? 96-well plate format for the initial screening as
described previously. Following the initial screening process selected dimers were
analysed further. Dimer constructs were expressed in TOPI OF' or HB2151 cells as
5 supernatants. Briefly, an individual colony from a freshly streaked plate was grown
overnight at 37°C in 2xTY with lOO^ig/ml ampicillin and 1% glucose. A 1/100 dilution
of this culture was inoculated into 2xTY with 100p,g/ml ampicillin and 0.1% glucose and
grown at 37°C shaking until OD600 was approximately 0.9. The culture was then induced
with ImM EPTG overnight at 30°C. The cells were removed by centrifugation and the
10 supernatant purified with protein A or L agarose.
Fab and cysteine hinge dimers were expressed as periplasmic proteins in HB2152 cells.
A 1/100 dilution of an overnight culture was inoculated into 2xTY with 0.1% glucose
and the appropriate antibiotics and grown at 30°C shaking until OD600 was
approximately 0.9. The culture was then induced with ImM IPTG for 3-4 hours at 25°C.
15 The cells were harvested by centrifugation and the pellet resuspended in periplasmic
preparation buffer (30mM Tris-HCl pH8.0, ImM EDTA, 20% sucrose). Following
centrifugation the supernatant was retained and the pellet resuspended in 5mM MgS04.
The supernatant was harvested again by centrifugation, pooled and purified.
20 1.6.2 Protein A/L purification
Optimisation of the purification of dimer proteins from Protein L agarose (Affitech,
Norway) or Protein A agarose (Sigma, UK) was examined. Protein was eluted by batch or
by column elution using a peristaltic pump. Three buffers were examined 0.1M
Phosphate-citrate buffer pH2.6, 0.2M Glycine pH2.5 and 0.1M Glycine pH2.5. The
25 optimal condition was determined to be under peristaltic pump conditions using 0.1M
Glycine pH2.5 over 10 column volumes. Purification from protein A was conducted
peristaltic pump conditions using 0.1M Glycine pH2.5.
1.6.3 FPLC purification
30 Further purification was carried out by FPLC analysis on the AKTA Explorer 100 system
(Amersham Biosciences Ltd). TAR1-5 and TAR1-5-19 dimers were fractionated by
cation exchange chromatography (1ml Resource S - Amersham Biosciences Ltd) eluted
with a 0-1M NaCl gradient in 50mM acetate buffer pH4. Hinge dimers were purified by
WO 2004/003019 PCT/GB2003/002804
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ion exchange (1ml Resource Q Amersham Biosciences Ltd) eluted with a 0-1M NaCl
gradient in 25mMTris HC1 pH 8.0. Fabs were purified by size exclusion chromatography
using a superose 12 (Amersham Biosciences Ltd ) column run at a flow rate of 0.5ml/min
in PBS with 0.05% tween. Following purification samples were concentrated using
5 vivaspin 5K cut off concentrators (Vivascience Ltd).
2.0 Results
2.1 TAR1-5 dimers
6 x 96 clones were picked from the round 2 selection encompassing all the libraries and
selection conditions. Supernatant preps were made and assayed by antigen and Protein L
ELISA, BIAcore and in the receptor assays. In ELISAs, positive binding clones were
identified from each selection method and were distributed between 3U, 5U and 7U
libraries. However, as the guiding dAb is always present it was not possible to
discriminate between high and low affinity binders by this method therefore BIAcore
analysis was conducted.
BIAcore analysis was conducted using the 2ml sup ernat ants. BIAcore analysis revealed
that the dimer Koff rates were vastly improved compared to monomelic TAR 1-5.
Monomer Koff rate was in the range of lO^M compared with dimer Koff rates which
- 20 were in the range of 10" 3 - 10" 4 M. 16 clones that appeared to have very slow off rates
were selected, these came from the 3U, 5U and 7U libraries and were sequenced. In
addition the supernatants were analysed for the ability to neutralise human TNFa in the
receptor assay.
25 6 lead clones (dl-d6 below) that neutralised in these assays and have been sequenced.
The results shows that out of the 6 clones obtained there are only 3 different second dAbs
(dAb 1 , dAb2 and dAb3) however where the second dAb is found more than once they are
linked with different length linkers.
30 TARl-5dl: 3U linker 2 nd &Ab=dAbl - ljj,g/ml Ag immunotube overnight wash
TARl-5d2: 3U linker 2 nd dAb=dAb2 - lfig/ml Ag immunotube overnight wash
TARl-5d3: 5U linker 2 nd dAb=dAb2 - ljag/ml Ag immunotube overnight wash
TARl-5d4: 5U linker 2 nd <±Ab=dAb3 - 20|ug/ml Ag immunotube overnight wash
10
15
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TARl~5d5: 5U linker 2 nd d Ab=d Ab 1 - 20|ag/ml Ag immunotube overnight wash
TARl-5d6: 7U linker 2 nd dAb=dAbl- Rl:l|j,g/ml Ag immunotube overnight wash,
R2:beads
The 6 lead clones were examined further. Protein was produced from the periplasm and
supernatant, purified with protein L agarose and examined in the cell and receptor assays.
The levels of neutralisation were variable (Table 1). The optimal conditions for protein
preparation were determined. Protein produced from HB2151 cells as supematants gave
the highest yield (approximately lOmgs/L of culture). The supematants were incubated
with protein L agarose for 2hrs at room temperature or overnight at 4°C. The beads were
washed with PBS/NaCl and packed onto an FPLC column using a peristaltic pump. The
beads were washed with 10 column volumes of PBS/NaCl and eluted with 0.1M glycine
pH2.5. In general, dimeric protein is eluted after the monomer.
TARl-5dl-6 dimers were purified by FPLC. Three species were obtained, by FPLC
purification and were identified by SDS PAGE. One species corresponds to monomer and
the other two species corresponds to dimers of different sizes. The larger of the two
species is possibly due to the presence of C terminal tags. These proteins were examined
in the receptor assay. The data presented in table 1 represents the optimum results
obtained from the two dimeric species (Figure 1 1)
The three second dAbs from the dimer pairs (ie, dAbl, dAb2 and dAb3) were cloned as
monomers and examined by ELISA and in the cell and receptor assay. All three dAbs
bind specifically to TNF by antigen ELISA and do not cross react with plastic or BSA. As
monomers, none of the dAbs neutralise in the cell or receptor assays.
2.1.2 TAR1-5-19 dimers
TAR1-5-19 was substituted for TAR1-5 in the 6 lead clones. Analysis of all TAR1-5-19
dimers in the cell and receptor assays was conducted using total protein (protein L
purified only) unless otherwise stated (Table 2). TARl-5-19d4 and TARl-5-19d3 have
the best ND50 (~5nM) in the cell assay, this is consistent with the receptor assay results
and is an improvement over TAJR1-5-19 monomer (ND 5 o~30nM). Although purified
TAR1-5 dimers give variable results in the receptor and cell assays TAR1-5-19 dimers
WO 2004/003019 PCT/GB2003/002804
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were more consistent. Variability was shown when using different elution buffers during
the protein purification. Elution using 0.1M Phosphate-citrate buffer pH2.6 or 0.2M
Glycine pH2.5 although removing all protein from the protein L agarose in most cases
rendered it less functional.
5
TARl-5-19d4 was expressed in the fermenter and purified on cation exchange FPLC to
yield a completely pure dimer. As with TARl-5d4 three species were obtained, by FPLC
purification corresponding to monomer and two dimer species. This dimer was amino
acid sequenced. TAR1-5-19 monomer and TARl-5-19d4 were then examined in the
10 receptor assay and the resulting IC50 for monomer was 30nM and for dimer was 8nM.
The results of the receptor assay comparing TAR1-5-19 monomer, TARl-5-19d4 and
TARl-5d4 is shown in figure 10.
TAR1-5-19 homodimers were made in the 3U, 5U and 7U vectors, expressed and purified
15 on Protein L. The proteins were examined in the cell and receptor assays and the resulting
IC 50 s (for receptor assay) and ND 50 s (for cell assay) were determined (table 3, figure 12).
2.2 Fabs
TAR1-5 and TAR1-5-19 dimers were also cloned into Fab format, expressed and purified
20 on protein L agarose. Fabs were assessed in the receptor assays (Table 4). The results
showed that for both TAR1-5-19 and TAR1-5 dimers the neutralisation levels were
similar to the original Gly 4 Ser linker dimers from which they were derived. A TAR1-5-19
Fab where TAR1-5-19 was displayed on both CH and CK was expressed, protein L
purified and assessed in the receptor assay. The resulting IC50 was approximately InM.
2.3 TAR1-27 dimers
3 x 96 clones were picked from the round 2 selection encompassing all the libraries and
selection conditions. 2ml supernatant preps were made for analysis in ELISA and
30 bioassays. Antigen ELISA gave 71 positive clones. The receptor assay of crude
supernatants yielded 42 clones with inhibitory properties (TNF binding 0-60%). In the
majority of cases inhibitory properties correlated with a strong ELISA signal. 42 clones
WO 2004/003019 PCT/GB2003/002804
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were sequenced, 39 of these have unique second dAb sequences. The 12 dimers that
gave the best inhibitory properties were analysed further.
The 12 neutralising clones were expressed as 200ml supernatant preps and purified on
protein L. These were assessed by protein L and antigen ELISA, BIAcore and in the
receptor assay. Strong positive ELISA signals were obtained in all cases. BIAcore
analysis revealed all clones to have fast on and off rates. The off rates were improved
compared to monomelic TAR1-27, however the off rate of TAR1-27 dimers was faster
(Koff is approximately in the range of 10" 1 and 10" 2 M) than the TAR1-5 dimers examined
previously (Koff is approximately in the range of 10~ 3 - 10" 4 M). The stability of the
purified dimers was questioned and therefore in order to improve stability, the addition on
5%glycerol, 0.5% Triton X100 or 0.5% NP40 (Sigma) was included in the purification of
2 TAR1-27 dimers (d2 and dl6). Addition of NP40 or Triton X100™ improved the yield
of purified product approximately 2 fold. Both dimers were assessed in the receptor
assay. TARl-27d2 gave IC50 of ~30nM under all purification conditions. TARl-27dl6
showed no neutralisation effect when purified without the use of stabilising agents but
gave an IC50 of ~50nM when purified under stabilising conditions. No further analysis
was conducted.
2.4 TAR2-5 dimers
3 x 96 clones were picked from the second round selections encompassing all the libraries
and selection conditions. 2ml supernatant preps were made for analysis. Protein A and
antigen ELISAs were conducted for each plate. 30 interesting clones were identified as
having good off-rates by BIAcore (Koff ranges between 10' 2 - 10" 3 M). The clones were
sequenced and 13 unique dimers were identified by sequence analysis.
Table 1: TAR1-5 dimers
Dimer
Cell
type
Purification
Protein
Fraction
Elution
conditions
Receptor/
Cell assay
TARl-5dl
HB2151
Protein L +
FPLC
small dimeric
species
0.1M glycine
pH2.5
RA~30nM
TARl-5d2
HB2151
Protein L +
FPLC
small dimeric
species
0.1M glycine
pH2.5
RA~50nM
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PCT/GB2003/002804
FPLC
species
pH2.5
M
TARl-5d3
HB2151
Protein L +
FPLC
large dimeric
species
0.1M glycine
pH2.5
RA-300
nM
TARl-5d4
HB2151
Protein L +
FPLC
small dimeric
species
0.1M glycine
pH2.5
RA~3n
M
TARl-5d5
HB2151
Protein L +
FPLC
large dimeric
species
0.1M glycine
pH2.5
RA-200
nM
TARl-5d6
HB2151
Protein L
+FPLC
Large dimeric
species
0.1M glycine
pH2.5
RA-100
nM
*note dimer 2 and dimer 3 have the same second dAb (called dAb2), however have
different linker lengths (d2 = (Gly 4 Ser) 3 , d3 - (Gly 4 Ser) 3 ). dAbl is the partner dAb to
dimers 1, 5 and 6. dAb3 is the partner dAb to dimer4. None of the partner dAbs
5 neutralise alone. FPLC purification is by cation exchange unless otherwise stated. The
optimal dimeric species for each dimer obtained by FPLC was determined in these
assays.
10 Table 2: TAR1-5-19 dimers
Dimer
Cell type
Purification
Protein
Fraction
Elution conditions
Recept
or/ Cell
assay
TARl-5-19dl
TOPI OF*
Protein L
Total protein
0.1M glycine pH 2.0
RA-15
nM
TAR1-5-19 d2 (no
stop codon)
TOPI OF'
Protein L
Total protein
0.1M glycine pH 2.0 +
0.05%NP40
RA~2n
M
TARl-5-19d3
(no stop codon)
TOPI OF'
Protein L
Total protein
0.1M glycine pH 2.5.+
0.05%NP40
RA~8n
M
TARl-5-19d4
TOPI OF'
Protein L +
FPLC
FPLC purified
fraction
0.1M glycine
pH2.0
RA~2-
5nM
CA-12
nM
TARl-5-19d5
TOPI OF'
Protein L
Total protein
0.1M glycine pH2.0 +
NP40
RA~8n
M
CA-10
nM
TARl-5-19d6
TOPI OF 5
Protein L
Total protein
0.1M glycine pH 2.0
RA-10
nM
Table 3: TAR1-5-19 homodimers
Dimer
Cell type
Purification
Protein Fraction
Elution conditions
Recept
or/ Cell
assay
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homodimer
nM
CA-30
nM
TAR1-5-19 5U
homodimer
HB2151
Protein L
Total protein
0.1M glycine pH2.5
RA~2n
M
M
TAR 1-5- 19 7U
homodimer
HB2151
1 ULdl piULClll
u.iivi glycine priz.3
nM
CA-15
nM
TAR1-5-19 cys
hinge
HB2151
Protein L + FPLC
FPLC • purified
dimer fraction
0.1M glycine pH2.5
RA~2n
M
TAR1-5-19CH/
TAR1-5-19 CK
HB2151
Protein
Total protein
0.1M glycine pH2.5
RA~ln
M
Table 4: TAR1-5/TAR1-5-19 Fabs
Dimer
Cell
type
Purification
Protein
Fraction
Elution
conditions
Rece
ptor/
Cell
assay
TAR1-5CH/
dAbl CK
HB2151
Protein L
Total protein
0,1M citrate pH2.6
RA-90
nM
TAR1-5CH/
dAb2 CK
HB2151
Protein L
Total protein
(MM glycine pH2.5
RA-30
nM
CA-60
nM
dAb3CH/
TAR1-5CK
HB2151
Protein L
Total protein
0.1M citrate pH2.6
RA-10
OnM
TAR1-5-19CH/
dAbl CK
HB2151
Protein L
Total protein
0.1M glycine pH2.0
RA~6n
M
dAbl CH/
TAR1-5-19CK
HB2151
Protein L
0.1M glycine
pH2.0
Myc/flag
RA~6n
M
TAR1-5-19CH/
dAb2 CK
HB2151
Protein L
Total protein
0.1M glycine pH2.0
RA~8n
M
CA-12
nM
TAR1-5-19CH/
dAb3CK
HB2151
Protein L
Total protein
0.1M glycine pH2.0
RA~3n
M
5
SUBSTITUTE SHEET (RULE 26)
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PCR construction of TAR1-5-19CYS dimer
See example 8 describing the dAb trimer. The trimer protocol gives rise to a mixture of
monomer, dimer and trimer.
5 Expression and purification of TAR1-5-19CYS dimer
The dimer was purified from the supernatant of the culture by capture on Protein L
agarose as outlined in the example 8.
Separation of TAR1-5-19CYS monomer from the TAR1-5-19CYS dimer
10 Prior to cation exchange separation, the mixed monomer/dimer sample was buffer
exchanged into 50 mM sodium acetate buffer pH 4.0 using a PD-10 column (Amersham
Pharmacia), following the manufacturer's guidelines. The sample was then applied to a
lmL Resource S cation exchange column (Amersham Pharmacia), which had been pre-
equilibrated with 50 mM sodium acetate pH 4.0. The monomer and dimer were separated
15 using the following salt gradient in 50 mM sodium acetate pH 4.0:
150 to 200 mM sodium chloride over 15 column volumes
200 to 450 mM sodium chloride over 10 column volumes
450 to 1000 mM sodium chloride over 15 column volumes
20
Fractions containing dimer only were identified using SDS-PAGE and then pooled and
the pH increased to 8 by the addition of 1/5 volume of 1M Tris pH 8.0.
In vitro functional binding assay: TNF receptor assay and cell assay
25 The affinity of the dimer for human TNFce was determined using the TNF receptor and
cell assay. IC50 in the receptor assay was approximately 0.3-0.8 nM; ND50 in the cell
assay was approximately 3-8 nM.
Other possible TAR1-5-19CYS dimer formats
30
PEG dimers and custom synthetic maleimide dimers
Nektar (Shearwater) offer a range of bi-maleimide PEGs [mPEG2-(MAL)2 or mPEG-
(MAL)2] which would allow the monomer to be formatted as a dimer, with a small linker
WO 2004/003019
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108
separating the dAbs and both being linked to a PEG ranging in size from 5 to 40 kDa. It
has been shown that the 5kDa mPEG-(MAL)2 (ie, [TARl-5-19]-Cys-maleimide-PEG x
2, wherein the maleimides are linked together in the dimer) has an affinity in the TNF
receptor assay of - 1-3 nM. Also the dimer can also be produced using TMEA (Tris[2-
5 maleimidoethyl] amine) (Pierce Biotechnology) or other bi- functional linkers.
It is also possible to produce the disulphide dimer using a chemical coupling procedure
using 2 3 2'-dithiodipyridine (Sigma Aldrich) and the reduced monomer.
10 Addition of a polypeptide linker or hinge to the C-terminus of the dAh.
A small linker, either (Gly4Ser) n where n=l to 10, eg, 1, 2, 3, 4, 5, 6 or 7, an
immunoglobulin (eg, IgG hinge region or random peptide sequence (eg, selected from a
library of random peptide sequences) can be engineered between the dAb and the terminal
cysteine residue. This can then be used to make dimers as outlined above.
15
Example 8
dAb trimerisation
20
Summary
For dAb trimerisation, a free cysteine is required at the C-terminus of the protein. The
cysteine residue, once reduced to give the free thiol, can then be used to specifically
25 couple the protein to a trimeric maleimide molecule, for example TMEA (Tris[2-
maleimido ethyl] amine) .
PGR construction of TAR1-5-19CYS
The following oligonucleotides were used to specifically PGR TAR1-5-19 with a Sail and
30 BamHL sites for cloning and also to introduce a C-terminal cysteine residue:
Sail
Trp Ser Ala Ser Thr Asp* He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val
35 1 TGG AGC GCG TCG ACG GAC ATC CAG ATG ACC CAG TCT CCA TCC TCT CTG TCT GCA TCT GTA
ACC TCG CGC AGC TGC CTG TAG GTC TAC TGG GTC AGA GGT AGG AGA GAC AGA CGT AGA CAT
Gly Asp Arg Val Thr He Thr Cys Arg Ala Ser Gin Ser He Asp Ser Tyr Leu His Trp
WO 2004/003019
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109
61 GGA GAC CGT GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC ATT GAT AGT TAT TTA CAT TGG
CCT CTG GCA CAG TGG TAG TGA ACG GCC CGT TCA GTC TCG TAA CTA TCA ATA AAT GTA ACC
Tyr
Gin
Gin
Lys
Pro
Gly
Lys
Ala
Pro
Lys
Leu
Leu
lie
Tyr
Ser
Ala
Ser
Glu
Leu
Gin
5
121
TAC
CAG
CAG
AAA
CCA
GGG
AAA
GCC
CCT
AAG
CTC
CTG
ATC
TAT
AGT
GCA
TCC
GAG
TTG
CAA
ATG
GTC
GTC
TTT
GGT
CCC
TTT
CGG
GGA
TTC
GAG
GAC
TAG
ATA
TCA
CGT
AGG
CTC
GTT
Ser
Gly
Val
Pro
Ser
Arg
Phe
Ser
Gly
Ser
Gly
Ser
Gly
Thr
Asp
Phe
Thr
Leu
Thr
lie
181
AGT
GGG
GTC
CCA
TCA
CGT
TTC
AGT
GGC
AGT
GGA
TCT
GGG
ACA
GAT
TTC
ACT
CTC
ACC
ATC
10
TCA
CCC
CAG
GGT
AGT
GCA
AAG
TCA
CCG
TCA
CCT
AGA
CCC
TGT
CTA
AAG
TGA
GAG
TGG
TAG
Ser
Ser
Leu
Gin
Pro
Glu
Asp
Phe
Ala
Thr
Tyr
Tyr
Cys
Gin
Gin
Val
Val
Trp
Arg
Pro
241
AGC
AGT
CTG
CAA
CCT
GAA
GAT
TTT
GCT
ACG
TAC
TAC
TGT
CAA
CAG
GTT
GTG
TGG
CGT
CCT
TCG
TCA
GAC
GTT
GGA
CTT
CTA
AAA
CGA
TGC
ATG
ATG
ACA
GTT
GTC
CAA
CAC
ACC
GCA
GGA
15
BamHI
Phe
Thr
Phe
Gly
Gin
Gly
Thr
Lys
Val
Glu
lie
Lys
Arg
Cys
***
** *
Gly
Ser
Gly
301
TTT
ACG
TTC
GGC
CAA
GGG
ACC
AAG
GTG
GAA
ATC
AAA
CGG
TGC
TAA
TAA
GGA
TCC
GGC
AAA
TGC
AAG
CCG
GTT
CCC
TGG
TTC
CAC
CTT
TAG
TTT
GCC
ACG
ATT
ATT
CCT
AGG
CCG
20
(* start ofTARl-5-19CYS sequence)
Forward primer
25 5 ' -TGGAGCGCGTCGACGGAC ATCC AGATGACCC AGTCTCCA-3 '
Reverse primer
5 ' -TTAGC AGCCGGATCCTTATT AGC ACCGTTTGATTTCC AC-3 5
30
The PGR reaction (50|liL volume) was set up as follows: 200|aM dNTPs, 0.4jliM of each
primer, 5 [xL of lOx P/wTurbo buffer (Stratagene), 100 ng of template plasmid (encoding
TAR1-5-19), l\xL of P/wTurbo enzyme (Stratagene) and the volume adjusted to 50jaL
using sterile water. The following PCR conditions were used: initial denaturing step 94
35 °C for 2 mins, then 25 cycles of 94 °C for 30 sees, 64 °C for 30 sec and 72 °C for 30 sec.
A final extension step was also included of 72 °C for 5 mins. The PCR product was
purified and digested with Sail and BamHI and ligated into the vector which had also
been cut with the same restriction enzymes. Correct clones were verified by DNA
sequencing.
40
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110
Expression and purification of TAR1-5-19CYS
TAR1-5-19CYS vector was transformed into BL21 (DE3) pLysS chemically competent
cells (Novagen) following the manufacturer's protocol. Cells carrying the dAb plasmid
were selected for using 100|Hg/mL carbenicillin and 37 \xg/mL chloramphenicol. Cultures
5 were set up in 2L baffled flasks containing 500 mL of terrific broth (Sigma- Aldrich),
100|ng/mL carbenicillin and 37 |xg/mL chloramphenicol. The cultures were grown at 30
°C at 200rpm to an O.D.600 of 1-1.5 and then induced with ImM DPTG (isopropyl-beta-
D-thiogalactopyranoside, from Melford Laboratories). The expression of the dAb was
allowed to continue for 12-16 hrs at 30 °C. It was found that most of the dAb was present
10 in the culture media. Therefore, the cells were separated from the media by centrifugation
(8,000xg* for 30 mins), and the supernatant used to purify the dAb. Per litre of
supernatant, 30 mL of Protein L agarose (Affitech) was added and the dAb allowed to
batch bind with stirring for 2 hours. The resin was then allowed to settle under gravity for
a further hour before the supernatant was siphoned off. The agarose was then packed into
15 a XK 50 column (Amersham Phamacia) and was washed with 10 column volumes of
PBS. The bound dAb was eluted with 100 mM glycine pH 2.0 and protein containing
fractions were then neutralized by the addition of 1/5 volume of 1 M Tris pH 8.0. Per litre
of culture supernatant 20 mg of pure protein was isolated, which contained a 50:50 ratio
of monomer to dimer.
20
Trimerisation of TAR1-5-19CYS
2.5 ml of 100 jjM TAR1-5-19CYS was reduce with 5 mM dithiothreitol and left at room
temperature for 20 minutes. The sample was then buffer exchanged using a PD-10
column (Amersham Pharmacia). The column had been pre-equilibrated with 5 mM
25 EDTA, 50 mM sodium phosphate pH 6.5, and the sample applied and eluted following
the manufactures guidelines. The sample was placed on ice until required. TMEA (Tris [2-
maleimido ethyl] amine) was purchased from Pierce Biotechnology. A 20 mM stock
solution of TMEA was made in 100% DMSO (dimethyl sulphoxide). It was found that a
concentration of TMEA greater than 3:1 (molar ratio of dAb:TMEA) caused the rapid
30 precipitation and cross-linking of the protein. Also the rate of precipitation and cross-
linking was greater as the pH increased. Therefore using 100 pM reduced TAR1-5-
19CYS, 25 |liM TMEA was added to trimerise the protein and the reaction allowed to
proceed at room temperature for two hours. It was found that the addition of additives
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such as glycerol or ethylene glycol to 20% (v/v), significantly reduced the precipitation
of the trimer as the coupling reaction proceeded. After coupling, SDS-PAGE analysis
showed the presence of monomer, dimer and trimer in solution.
5 Purification of the trimeric TAR1-5-19CYS
40 jaL of 40% glacial acetic acid was added per mL of the TMEA-TAR1 - 5-1 9cys reaction
to reduce the pH to -4. The sample was then applied to a lmL Resource S cation
exchange column (Amersham Pharmacia), which had been pre-equilibrated with 50 niM
sodium acetate pH 4.0. The dimer and trimer were partially separated using a salt gradient
10 of 340 to 450 mM Sodium chloride, 50 mM sodium acetate pH 4.0 over 30 column
volumes. Fractions containing trimer only were identified using SDS-PAGE and then
pooled and the pH increased to 8 by the addition of 1/5 volume of 1M Tris pH 8.0. To
prevent precipitation of the trimer during concentration steps (using 5K cut off Viva spin
concentrators; Vivascience), 10% glycerol was added to the sample.
15
In vitro functional binding assay: TNF receptor assay and cell assay
The affinity of the trimer for human TNFce was determined using the TNF receptor and
cell assay. IC50 in the receptor assay was 0.3nM; ND50 in the cell assay was in the range
of 3 to lOnM (eg, 3nM).
20
Other possible TAR1-5-19CYS trimer formats
. TAR1-5-19CYS may also be formatted into a trimer using the following reagents:
PEG trimers and custom synthetic maleimide trimers
25 Nektar (Shearwater) offer a range of multi arm PEGs, which can be chemically modified
at the terminal end of the PEG. Therefore using a PEG trimer with a maleimide functional
group at the end of each arm would allow the trimerisation of the dAb in a manner similar
to that outlined above using TMEA. The PEG may also have the advantage in increasing
the solubility of the trimer thus preventing the problem of aggregation. Thus, one could
30 produce a dAb trimer in which each dAb has a C-terminal cysteine that is linked to a
maleimide functional group, the maleimide functional groups being linked to a PEG
trimer.
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Addition of a polypeptide linker or hinge to the C-terminus of the dAb
A small linker, either (Gly 4 Ser) n where n= 1 to 10, eg, 1, 2, 3, 4, 5, 6 or 7 , an
immunoglobulin (eg, IgG hinge region or random peptide sequence (eg, selected from a
library of random peptide sequences) could be engineered between the dAb and the
5 terminal cysteine residue. When used to make multimers (eg, dimers or trimers), this
again would introduce a greater degree of flexibility and distance between the individual
monomers, which may improve the binding characteristics to the target, eg a multisubunit
target such as human TNFce.
10 Example 9.
Selection of a collection of single domain antibodies (dAbs) directed against human
serum albumin (HSA) and mouse serum albumin (MSA).
15 This example explains a method for making a single domain antibody (dAb) directed
against serum albumin. Selection of dAbs against both mouse serum albumin (MSA) and
human serum albumin (HSA) is described. Three human phage display antibody libraries
were used in this experiment, each based on a single human framework for V H (see
Figure 13: sequence of dummy V H based on V3-23/DP47 and JH4b) or Vk (see Figure
20 15: sequence of dummy Vk based on ol2/o2/DPK9 and Jkl) with side chain diversity
encoded by NNK codons incorporated in complementarity determining regions (CDR1 ,
CDR2 and CDR3).
Library 1 (Vh):
25 Diversity at positions: H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95,
H97, H98.
Library size: 6.2 x 10 9
Library 2 (Vh):
30 Diversity at positions: H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95,
H97, H98, H99, H100, HlOOa, H100b.
Library size: 4.3 x 10 9
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Library 3 (Vk):
Diversity at positions: L30, L31, L32, L34, L50, L53, L91, L92, L93, L94, L96
Library size: 2 x 10 9
The Vh and Vk libraries have been preselected for binding to generic ligands protein A
and protein L respectively so that the majority of clones in the unselected libraries are
functional. The sizes of the libraries shown above correspond to the sizes after
preselection.
Two rounds of selection were performed on serum albumin using each of the libraries
separately. For each selection, antigen was coated on immunotube (nunc) in 4ml of PBS
at a concentration of 100/ig/ml. In the first round of selection, each of the three libraries
was panned separately against HSA (Sigma) and MSA (Sigma). In the second round of
selection, phage from each of the six first round selections was p aimed against (i) the
same antigen again (eg 1 st round MSA, 2 nd round MSA) and (ii) against the reciprocal
antigen (eg 1 st round MSA, 2 nd round HSA) resulting in a total of twelve 2 nd round
selections. In each case, after the second round of selection 48 clones were tested for
binding to HSA and MSA. Soluble dAb fragments were produced as described for scFv
fragments by Harrison et al, Methods Enzymol. 1996;267:83-109 and standard ELISA
protocol was followed (Hoogenboom et ah (1991) Nucleic Acids Res., 19: 4133) except
that 2% tween PBS was used as a blocking buffer and bound dAbs were detected with
either protein L-HRP (Sigma) (for the V/cs) and protein A -HRP (Amersham Pharmacia
Biotech) (for the Vhs).
dAbs that gave a signal above background indicating binding to MSA, HSA or both were
tested in ELISA insoluble form for binding to plastic alone but all were specific for serum
albumin. Clones were then sequenced (see table below) revealing that 21 unique dAb
sequences had been identified. The minimum similarity (at the amino acid level) between
the Vk dAb clones selected was 86.25% ((69/80)xl00; the result when all the diversified
residues are different, eg clones 24 and 34). The minimum similarity between the V H dAb
clones selected was 94 % ((127/136)xl00).
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Next, the serum albumin binding dAbs were tested for their ability to capture
biotinylated antigen from solution. ELISA protocol (as above) was followed except that
ELIS A plate was coated with 1 /ig/ml protein L (for the V/c clones) and 1 jug/ml protein A
(for the Vh clones). Soluble dAb was captured from solution as in the protocol and
5 detection was with biotinylated MSA or HSA and streptavidin HRP. The biotinylated
MSA and HSA had been prepared according to the manufacturer's instructions, with the
aim of achieving an average of 2 biotins per serum albumin molecule. Twenty four
clones were identified that captured biotinylated MSA from solution in the ELISA. Two
of these (clones 2 and 38 below) also captured biotinylated HSA. Next, the dAbs were
10 tested for their ability to bind MSA coated on a CMS biacore chip. Eight clones were
found that bound MSA on the biacore.
dAb (all
capture
biotinylated
MSA)
V/c library 3
template
(dummy)
2, 4, 7, 41,
38, 54
46, 47, 52, 56
13,15
30,35
19,
22,
23,
24,
31,
33,
34,
53,
H>
12,
17,
18,
16, 21
25, 26
27,
55,
V H library 1
(and 2)
template
(dummy)
8,10
36,
H
or k CDR1
CDR2
CDR3
Binds
MSA Captures
in biotinylated
biacore? HSA?
K
XXXLX
XASXLQS
QQXXXXPXT
K
SSYLN
RASPLQS
QQTYSVPPT
K
SSYLN
RASPLQS
QQTYRIPPT
K
FKSLK
NASYLQS
QQWYWPVT
K
YYHLK
KASTLQS
QQVRKVPRT
K
RRYLK
QASVLQS
QQGLYPPIT
K
YNWLK
RASSLQS
QQNWIPRT
K
LWHLR
HASLLQS
QQSAVYPKT
K
FRYLA
HASHLQS
QQRLLYPKT
K
FYHLA
PASKLQS
QQRARWPRT
K
IWHLN
RASRLQS
QQVARVPRT
K
YRYLR
KASSLQS
QQYVGYPRT
K
LKYLK
NASHLQS
QQTTYYPIT
K
LRYLR
KASWLQS
QQVLYYPQT
K
LRSLK
AASRLQS
QQWYWPAT
K
FRHLK
AASRLQS
QQVALYPKT
V
K
RKYLR
TASSLQS
QQNLFWPRT
S
K
RRYLN
AASSLQS
QQMLFYPKT
K
IKHLK
GASRLQS
QQGARWPQT
s
K
YYHLK
KASTLQS
QQVRKVPRT
K
YKHLK
NASHLQS
QQVGRYPKT
K
FKSLK
NASYLQS
QQWYWPVT
H
XXYXXX
XI XXXGXXTXYAD SVKG
XXXX(XXXX) FDY
H
WVYQMD
S I S AFGAKTLYADS VKG
LSGKFDY
H
WSYQMT
SISSFGSSTLYADSVKG
GRDHNYSLFDY
S all 4 bind
S both bind
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In all cases the frameworks were identical to the frameworks in the corresponding
dummy sequence, with diversity in the CDRs as indicated in the table above.
Of the eight clones that bound MSA on the biacore, two clones that are highly expressed
5 in E. coli (clones MSA16 and MSA26) were chosen for further study (see example 10).
Full nucleotide and amino acid sequences for MSA16 and 26 are given in figure 16.
Example 10.
10 Determination of affinity and serum half-life in mouse of MSA binding dAbs MSA16
and MSA26.
dAbs MSA16 and MSA26 were expressed in the periplasm oiE. coli and purified using
batch absorbtion to protein L-agarose affinity resin (Affitech, Norway) followed by
15 elution with glycine at pH 2.2. The purified dAbs were then analysed by inhibition
biacore to determine K d . Briefly, purified MSA16 and MSA26 were tested to determine
the concentration of dAb required to achieve 200RUs of response on a biacore CMS chip
coated with a high density of MSA. Once the required concentrations of dAb had been
determined, MSA antigen at a range of concentrations around the expected Kd was
20 premixed with the dAb and incubated overnight. Binding to the MSA coated biacore chip
of dAb in each of the premixes was then measured at a high flow-rate of 30 jul/minute.
The resulting curves were used to create Klotz plots, which gave an estimated Kd of
200nM for MSA16 and 70nM for MSA 26 (Figure 17 A & B).
25 Next, clones MSA16 and MSA26 were cloned into an expression vector with the HA tag
(nucleic acid sequence: TATCCTTATGATGTTCCTGATTATGCA and amino acid
sequence: YPYDVPDYA) and 2-10 mg quantities were expressed in E. coli and purified
from the supernatant with protein L-agarose affinity resin (Affitech, Norway) and eluted
with glycine at pH2.2. Serum half life of the dAbs was determined in mouse. MSA26
30 and MSA16 were dosed as single i.v. injections at approx 1.5mg/kg into CD1 mice.
Analysis of serum levels was by goat anti-HA (Abeam, UK) capture and protein L-HRP
(invitrogen) detection ELISA which was blocked with 4% Marvel. Washing was with
0.05% tween PBS. Standard curves of known concentrations of dAb were set up in the
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presence of lxmouse serum to ensure comparability with the test samples. Modelling
with a 2 compartment model showed MSA-26 had a tl/2ce of 0.16hr ? a tl/2jS of 14.5hr and
an area under the curve (AUC) of 465hr.mg/ml (data not shown) and MSA- 16 had a tl/2a
of 0.98hr, a tl/2/3 of 36.5hr and an AUC of 913hr.mg/ml (figure 18). Both anti-MSA
5 clones had considerably lengthened half life compared with HEL4 (an anti-hen egg white
lysozymedAb) which had a tl/2a of 0.06hr> and a tl/2(3 of 0.34hr.
Example 11.
10 Creation of V h -Vh and Vk- Vk dual specific Fab like fragments
This example describes a method for making V H ~ Vh and Vk-Vk dual specifics as Fab
like fragments. Before constructing each of the Fab like fragments described, dAbs that
bind to targets of choice were first selected from dAb libraries similar to those described
15 in example 9. A V H dAb, HEL4, that binds to hen egg lysozyme (Sigma) was isolated
and a second Vh dAb (TAR2h-5) that binds to TNFa receptor (R and D systems) was also
isolated. The sequences of these are given in the sequence listing. A Vk dAb that binds
TNFa; (TAR 1-5 -19) was isolated by selection and affinity maturation and the sequence is
also set forth in the sequence listing. A second Vk dAb (MSA 26) described in example 9
20 whose sequence is in figure 1 7B was also used in these experiments.
DNA from expression vectors containing the four dAbs described above was digested
with enzymes Sail and NotI to excise the DNA coding for the dAb. A band of the
expected size (300-400bp) was purified by running the digest on an agarose gel and
25 excising the band, followed by gel purification using the Qiagen gel purification kit
(Qiagen, UK). The DNA coding for the dAbs was then inserted into either the Ch or Ck
vectors (Figs 8 and 9) as indicated in the table below.
dAb
Target antigen
dAb V H or
dAb Vk
Inserted into
vector
tag(C
terminal)
Antibiotic
resisitance
HEL4
Hen egg lysozyme
V h
Ch
myc
Chloramphenicol
TAR2-5
TNF receptor
V h
Ck
flag
Ampicillin
TAR1-S-19
TNF a
Vk
C h
myc
Chloramphenicol
MSA 26
Mouse serum albumin
Vk
Ck
flag
Ampicillin
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The V H C h and V H Ck constructs were cotransformed into HB2151 cells. Separately, the
V/c C H and Vk Ck constructs were cotransformed into HB2151 cells. Cultures of each of
the cotransformed cell lines were grown overnight (in 2xTy containing 5% glucose,
10/xg/ml chloramphenicol and 100/xg/ml ampicillin to maintain antibiotic selection for
5 both C H and Ck plasmids). The overnight cultures were used to inoculate fresh media
(2xTy, 10/xg/ml chloramphenicol and 100/xg/ml ampicillin) and grown to OD 0.7-0.9
before induction by the addition of IPTG to express their C H and Ck constructs.
Expressed Fab like fragment was then purified from the periplasm by protein A
purification (for the contransformed V H C H and V H Ck) and MSA affinity resin
10 purification (for the contransformed Vk Ch and Vk Ck).
Vh-Vh dual specific
Expression of the V H Ch and V H Ck dual specific was tested by running the protein on a
gel. The gel was blotted and a band the expected size for the Fab fragment could be
15 detected on the Western blot via both the myc tag and the flag tag, indicating that both the
V H C h and V H Ck parts of the Fab like fragment were present. Next, in order to
determine whether the two halves of the dual specific were present in the same Fab-like
fragment, an ELISA plate was coated overnight at 4°C with 100 /xl per well of hen egg
lysozyme (HEL) at 3 mg/ml in sodium bicarbonate buffer. The plate was then blocked
20 (as described in example 1) with 2% tween PBS followed by incubation with the V H C H
/V H Ck dual specific Fab like fragment. Detection of binding of the dual specific to the
HEL was via the non cognate chain using 9el0 (a monoclonal antibody that binds the
myc tag, Roche) and anti mouse IgG-HRP (Amersham Pharmacia Biotech). The signal
for the V H C h /V h Ck dual specific Fab like fragment was 0.154 compared to a
25 background signal of 0.069 for the V H Ck chain expressed alone. This demonstrates that
the Fab like fragment has binding specificity for target antigen.
V K -V K dual specific
After purifying the contransformed Vk C h and Vk Ck dual specific Fab like fragment on
30 an MSA affinity resin, the resulting protein was used to probe an ELISA plate coated with
1/xg/ml TNFa and an ELISA plate coated with 10/xg/ml MSA. As predicted, there was
signal above background when detected with protein L-HRP on bot ELISA plates (data
not shown). This indicated that the fraction of protein able to bind to MSA (and therefore
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purified on the MSA affinity column) was also able to bind TNFa in a subsequent
ELISA, confirming the dual specificity of the antibody fragment. This fraction of protein
was then used for two subsequent experiments. Firstly, an ELISA plate coated with
, 1/xg/ml TNFa was probed with dual specific Vtc C H and Vk Ck Fab like fragment and
5 also with a control TNFa binding dAb at a concentration calculated to give a similar
signal on the ELISA. Both the dual specific and control dAb were used to probe the
ELISA plate in the presence and in the absence of 2mg/ml MSA. The signal in the dual
specific well was reduced by more than 50% but the signal in the dAb well was not
reduced at all (see figure 19a). The same protein was also put into the receptor assay with
10 and without MSA and competition by MSA was also shown (see figure 19c). This
demonstrates that binding of MSA to the dual specific is competitive with binding to
TNFo;.
15 Example 12.
Creation of a Vk- Vic dual specific cys bonded dual specific with specificity for
mouse serum albumin and TNFa
20 This example describes a method for making a dual specific antibody fragment specific
for both mouse serum albumin and TNFo; by chemical coupling via a disulphide bond.
Both MSA16 (from example 1) and TAR1-5-19 dAbs were recloned into a pET based
vector with a C terminal cysteine and no tags. The two dAbs were expressed at 4-10 mg
levels and purified from the supernatant using protein L-agarose affinity resin (Affitiech,
25 Norway). The cysteine tagged dAbs were then reduced with dithiothreitol. The TAR1-5-
1 9 dAb was then coupled with dithiodipyridine to block reformation of disulphide bonds
resulting in the formation of PEP 1-5-19 homodimers. The two different dAbs were then
mixed at pH 6.5 to promote disulphide bond formation and the generation of TAR1-5-19,
MSA16 cys bonded heterodimers. This method for producing conjugates of two unlike
30 proteins was originally described by Bang et al (King TP, Li Y Kochoumian L
Biochemistry. 1978 voll7: 1499-506 Preparation of protein conjugates via intermolecular
disulfide bond formation.) Heterodimers were separated from monomelic species by
cation exchange. Separation was confirmed by the presence of a band of the expected
WO 2004/003019 PCT/GB2003/002804
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size on a SDS gel. The resulting heterodimeric species was tested in the TNF receptor
assay and found to have an IC50 for neutralising TNF of approximately 18 nM. Next,
the receptor assay was repeated with a constant concentration of heterodimer (18nM) and
a dilution series of MSA and HSA. The presence of HSA at a range of concentrations (up
5 to 2 mg/ml) did not cause a reduction in the ability of the dimer to inhibit TNFo? .
However, the addition of MSA caused a dose dependant reduction in the ability of the
dimer to inhibit TNFo: (figure 20).This demonstrates that MSA and TNFa compete for
binding to the cys bonded TAR1-5-19, MSA16 dimer.
10 Data Summary
A summary of data obtained in the experiments set forth in the foregoing examples is set
forth in Annex 4.
All publications mentioned in the present specification, and references cited in said
15 publications, are herein incorporated by reference. Various modifications and variations
of the described methods and system of the invention will be apparent to those skilled in
the art without departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred embodiments, it
should be understood that the invention as claimed should not be unduly limited to such
20 specific embodiments. Indeed, various modifications of the described modes for carrying
out the invention which are obvious to those skilled in molecular biology or related fields
are intended to be within the scope of the following claims.
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Annex 1; polypeptides which enhance half-life in vivo.
Alpha- 1 Glycoprotein (Orosomucoid) (AAG)
Alpha- 1 Antichyromotrypsin (ACT)
Alpha- 1 Antitrypsin (AAT)
Alpha- 1 Microglobulin (Protein HC) (AIM)
Alpha-2 Macroglobulin (A2M)
Antithrornbin III (AT III)
Apolipoprotein A-l (Apo A-l)
Apoliprotein B (Apo B)
Beta-2-microglobulin (B2M)
Ceruloplasmin (Cp)
Complement Component (C3)
Complement Component (C4)
CI Esterase Inhibitor (CI INH)
C-Reactive Protein (CRP)
Cystatin C (Cys C)
Ferritin (FER)
Fibrinogen (FIB)
Fibronectin (FN)
Haptoglobin (Hp)
Hemopexin (HPX)
Immunoglobulin A (IgA)
Immunoglobulin D (IgD)
Immunoglobulin E (IgE)
Immunoglobulin G (IgG)
Immunoglobulin M (IgM)
Immunoglobulin Light Chains (kapa/lambda)
Lipoprotein(a) [Lp(a)]
Mannose-bindign protein (MBP)
Myoglobin (Myo)
Plasminogen (PSM)
Prealbumin (Transthyretin) (PAL)
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Retinol-binding protein (RBP)
Rheomatoid Factor (RF)
Serum Amyloid A (SAA)
Soluble Tranferrin Receptor (sTfR)
5 Transferrin (Tf)
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Annex 2
Pairing
Therapeutic relevant references.
TNF
ALPHA/TGF-p
• TGF-b and TNF when injected into the ankle joint of collagen induced
arthritis model significantly enhanced joint inflammation. In non-collagen
challenged mice there was no effect.
TNF ALPHA/IL-
1
• TNF and IL-1 synergize in the pathology of uveitis.
• TNF and IL-1 synergize in the pathology of malaria (hypoglycaemia, NO).
• TNF and IL-1 synergize in the induction of polymorphonuclear (PMN)
cells migration in inflammation.
• IL-1 and TNF synergize to induce PMN infiltration into the peritoneum.
• IL-1 and TNF synergize to induce the secretion of IL-1 by endothelial cells.
Important in inflammation.
• IL-1 or TNF alone induced some cellular infiltration into knee synovium.
IL-1 induced PMNs, TNF - monocytes. Together they induced a more
severe infiltration due to increased PMNs.
• Circulating myocardial depressant substance (present in sepsis) is low
levels of IL-1 andTNFacting synergistically.
TNF ALPHA/IL-2
• Most relating to synergistic activation of killer T-cells.
TNF ALPHA/IL-3
• Synergy of interleukin 3 and tumor necrosis factor alpha in stimulating
clonal growth of acute myelogenous leukemia blasts is the result of
induction of secondary hematopoietic cytokines by tumor necrosis factor
alpha.
• Cancer Res. 1992 Apr 15;52(8):2 197-201.
TNF ALPHA/IL-4
• IL-4 and TNF svnergize to induce VCAM expression on endothelial cells.
Implied to have a role in asthma. Same for synovium - implicated in RA.
• TNF and IL-4 synergize to induce IL-6 expression in keratinocytes.
• Sustained elevated levels of VCAM- 1 in cultured fibroblast-like
synoviocytes can be achieved by TNF-alpha in combination with either IL-
4 or IL-1 3 through increased mRNA stability. Am J Pathol. 1999
Apr;154(4):l 149-58
TNF ALPHA/IL-5
• Relationship between the tumor necrosis factor system and the serum
mterleukin-4, interleukin-5 9 interleukin- 8, eosinophil cationic protein, and
immunoglobulin E levels in the bronchial hyperreactivity of adults and
their children. Allergy Asthma Proc. 2003 Mar-Apr;24(2): 111-8.
TNF ALPHA/IL-6
• TNF and IL-6 are potent growth factors for OH-2, a novel human myeloma
ST C
cell line. Ear J Haematol 1994 Jul;53(l):3 1-7.
TNF ALPHA/IL-8
• TNF and IL-8 synergized with PMNs to activate platelets. Implicated in
Acute Respiratory Distress Syndrome.
• See IL-5/TNF (asthma). Synergism between interleukin-8 and tumor
necrosis factor-alpha for neutrophil-mediated platelet activation. Eur
Cytokine Netw. 1994 Sep-Oct;5(5):455-60. (adult respiratory distress
syndrome (ARDS))
TNF ALPHA/IL-9
TNF ALPHA/IL-
10
• IL-10 induces and synergizes with TNF in the induction of HIV expression
in chronically infected T-cells.
TNF ALPHA/IL-
11
• Cytokines synergistically induce osteoclast differentiation: support by
immortalized or normal calvarial cells. Am J Physiol Cell Physiol 2002
Sep;283(3):C679-87. (Bone loss)
TNF ALPHA/IL-
12
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TNF ALPHA/IL-
13
• Sustained elevated levels of VCAM-1 in cultured fibroblast-like
synoviocytes can be achieved by TNF-alpha in combination with either IL-
4 or IL-13 through increased rnRNA stability. Am J Pathol. 1999
Apr;154(4):l 149-58.
• Interleukin-13 and tumour necrosis factor-alpha synergistically induce
eotaxin production in human nasal fibroblasts. Clin Exp Allergy. 2000
Mar;30(3):348-55.
• Interleukin-13 and tumour necrosis factor-alpha synergistically induce
eotaxin production in human nasal fibroblasts. Clin Exp Allergy. 2000
Mar;30(3):348-55 (allergic inflammation)
• Implications of serum TNF -beta and IL-13 in the treatment response of
childhood nephrotic syndrome. Cytokine. 2003 Feb 7;21(3):155-9.
TNF ALPHA/IL-
14
• Effects of inhaled tumour necrosis factor alpha in subjects with mild
asthma. Thorax. 2002 Sep;57(9):774-8.
TNF ALPHA/IL-
15
• Effects of inhaled tumour necrosis factor alpha in subjects with mild
asthma. Thorax. 2002 Sep;57(9):774-8.
TNF ALPHA/IL-
16
• Tumor necrosis factor-alpha-induced synthesis of interleukin-16 in airway
epithelial cells: priming for serotonin stimulation. Am J Respir Cell Mo I
Biol. 2003 Mar;28(3):354-62. (airway inflammation)
• Correlation of circulating interleukin 1 6 with proinflammatory cytokines in
patients with rheumatoid arthritis. Rheumatology (Oxford). 2001
Apr;40(4):474-5. No abstract available.
• Interleukin 1 6 is up-regulated in Crohn's disease and participates in TNBS
colitis in mice. Gastroenterology. 2000 Oct;119(4):972-82.
TNF ALPHA/IL-
17
• Inhibition of interleukin- 17 prevents the development of arthritis in
vaccinated mice challenged with Borrelia burgdorferi. Infect Immun. 2003
Jun;71(6):3437-42.
• Interleukin 17 synergises with tumour necrosis factor alpha to induce
cartilage destruction in vitro. Ann Rheum Dis. 2002 Oct;61(10):870-6.
• A role of GM-CSF in the accumulation of neutrophils in the airways caused
byIL-17 and TNF-alpha. Eur Respir J. 2003 Mar;21(3):387-93. (Airway
inflammation)
• Abstract Interleukin- 1, tumor necrosis factor alpha, and interleukin- 17
synergistically up-reeulate nitric oxide and prostaglandin E2 production in
explants of human osteoarthritic knee menisci. Arthritis Rheum. 2001
Sep;44(9):2078-83.
TNF ALPHA/IL-
18
• Association of interleukin- 18 expression with enhanced levels of both
interleukin- lbeta and tumor necrosis factor alpha in knee synovial tissue of
patients with rheumatoid arthritis. Arthritis Rheum. 2003 Feb;48(2):339-
47.
• Abstract Elevated levels of interleukin- 1 8 and tumor necrosis factor-alpha
in serum of patients with type 2 diabetes mellitus: relationship with diabetic
nephropathy. Metabolism. 2003 May;52(5): 605-8.
TNF ALPHA/IL-
19
• Abstract IL-19 induces production of IL-6 and TNF-alpha and results in
cell apoptosis through TNF-alpha. J Immunol. 2002 Oct 15;169(8):4288-
97.
TNF ALPHA/IL-
20
• Abstract Cytokines: IL-20 - a new effector in skin inflammation. Curr Biol.
2001 Jull0;ll(13):R531-4
TNF
ALPHA/Complem
ent
• Inflammation and coagulation: implications for the septic patient. Clin
Infect Dis. 2003 May 15;36(10): 1259-65. Epub 2003 May 08. Review.
TNF
ALPHA/IFN-y
• MHC induction in the brain.
• Synergize in anti- viral response/IFN-p induction.
• Neutrophil activation/ respiratory burst.
• Endothelial cell activation
• Toxicities noted when patients treated with TNF/IFN-y as anti-viral therapy
WO 2004/003019
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• Fractalkine expression by human astrocytes.
• Many papers on inflammatory responses - i.e. LPS, also macrophage
activation.
• Anti-TNF and anti-IFN-y synergize to protect mice from lethal
endotoxemia.
TGF-p/IL-1
• Prostaglndin synthesis by osteoblasts
• IL-6 production by intestinal epithelial cells (inflammation model)
• Stimulates IL-1 1 and IL-6 in lung fibroblasts (inflammation model)
• IL-6 and IL-8 production in the retina
TGF-p/IL-6
• Chondrocarcoma proliferation
IL-l/IL-2
• B-cell activation
• LAK cell activation
• T-cell activation
• IL-1 synergy with IL-2 in the generation of lymphokine activated killer
cells is mediated by TNF-alpha and beta (lymphotoxin). Cytokine. 1992
Nov;4(6):479-87.
IL-l/IL-3
IL-l/IL-4
• B-cell activation
• IL-4 induces IL-1 expression in endothelial cell activation.
IL-l/IL-5
IL-l/IL-6
• B cell activation
• T cell activation (can replace accessory cells)
• IL-1 induces IL-6 expression
JL
• C3 and serum amyloid expression (acute phase response)
• HIV expression
• Cartilage collagen breakdown.
IL-l/IL-7
• IL-7 is requisite for IL-1 -induced thymocyte proliferation. Involvement of
IL-7 in the synergistic effects of granulocyte-macrophage colony-
stimulating factor or tumor necrosis factor with IL-1. J Immunol. 1992 Jan
1;148(1):99-105.
IL-l/IL-8
IL-l/IL-10
TT _1/TT _1 1
• (~VtnlHnp<! <?vnerffi stic all v induce osteoclast differentiation: sutTDortbv
immortalized or normal calvarial cells. Am J Physiol Cell Physiol 2002
Sep;283(3):C679-87. (Bone loss)
IL-l/IL-16
• Correlation of circulating interleukin 1 6 with proinflammatorv cytokines in
patients with rheumatoid arthritis. Rheumatology (Oxford). 2001
Apr;40(4):474-5. No abstract available.
IL-l/IL-17
• Inhibition of interleukin- 17 prevents the development of arthritis in
vaccinated mice challenged with Borrelia burgdorferi. Infect Immun. 2003
Jun;71(6):3437-42.
• Contribution of interleukin 17 to human cartilage degradation and synovial
inflammation in osteoarthritis. Osteoarthritis Cartilage. 2002
Oct;10(10):799-807.
• Abstract Interleukin- 1, tumor necrosis factor alpha, and interleukin- 17
synergistically up-regulate nitric oxide and prostaglandin E2 production in
explants of human osteoarthritic knee menisci. Arthritis Rheum. 2001
Sep;44(9):2078-83.
IL-l/IL-18
• Association of interleukin- 1 8 expression with enhanced levels of both
interleukin- lbeta and tumor necrosis factor alpha in knee synovial tissue of
patients with rheumatoid arthritis. Arthritis Rheum. 2003 Feb;48(2):339-47.
IL-l/IFN-g
IL-2/IL-3
• T-cell proliferation
• B cell proliferation
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IL-2/IL-4
• B~cell proliferation
• T-cell proliferation
• (selectively inducing activation of CD8 and NK lymphocytes)IL-2R beta
agonist Pl-30 acts in synergy with IL-2, IL-4, IL-9, and IL-15: biological
and molecular effects. J Immunol 2000 Oct 15;165(8): 4312-8.
IL-2/IL-5
• B-cell proliferation/ Ig secretion
• IL-5 induces IL-2 receptors on B-cells
IL-2/IL-6
• Development of cytotoxic T-cells
IL-2/IL-7
IL-2/IL-9
• See IL-2/IL-4 (NK-cells)
IL-2/IL-10
• B-cell activation
IL-2/IL-12
• IL-12 synergizes with IL-2 to induce lymphokine- activated cytotoxicity
and perforin and granzyme gene expression in fresh human NK cells. Cell
Immunol 1995 Oct l;165(l):33-43. (T-cell activation)
IL-2/IL-15
• See IL-2/IL-4 (NK cells)
• (T cell activation and proliferation) IL-15 and IL-2: a matter of life and
death for T cells in vivo. Nat Med. 2001 Jan;7(l): 114-8.
IL-2/IL-16
• Synergistic activation of CD4+ T cells by IL-16 and IL-2. J Immunol 1998
Mar 1;160(5):2115-20.
IL-2/IL-17
• Evidence for the early involvement of interleukin 17 in human and
experimental renal allograft rejection. J Pathol 2002 Jul; 197(3): 3 22-3 2.
IL-2/IL-18
• Interleukin 18 (IL-18) in synergy with IL-2 induces lethal lung injury in
mice: a potential role for cytokines, chemokines, and natural killer cells in
the pathogenesis of interstitial pneumonia. Blood. 2002 Feb 15;99(4):1289-
98.
IL-2/TGF-p
• Control of CD4 effector fate: transforming growth factor beta 1 and
interleukin 2 synergize to prevent apoptosis and promote effector
expansion. J Exp Med. 1995 Sep l;182(3):699-709.
IL-2/IFN-y
• Ig secretion by B-cells
• IL-2 induces IFN-y expression by T-cells
IL-2/IFN-cc/|3
• None
IL-3/IL-4
• Synergize in mast cell growth
• Synergistic effects of IL-4 and either GM-CSF or IL-3 on the induction of
CD23 expression by human monocytes: regulatory effects of IFN-alpha and
IFN-gamma. Cytokine. 1994 Jul;6(4):407-13.
IL-3/IL-5
IL-3/IL-6
TT -3/IFN-v
• IL-4 and IFN-gamma synergistically increase total polymeric IgA receptor
levels in human intestinal epithelial cells. Role of protein tyrosine kinases.
J Immunol 1996 Jun 15;156(12):4807-14.
IL-3/GM-CSF
• Differential regulation of human eosinophil IL-3, IL-5, and GM-CSF
receptor alpha-chain expression by cytokines: IL-3, IL-5, and GM-CSF
down-regulate IL-5 receptor alpha expression with loss of IL-5
responsiveness, but up-regulate IL-3 receptor alpha expression. J Immunol
2003 Jun l;170(ll):5359-66. (allergic inflammation)
IL-4/IL-2
• IL-4 synergistically enhances both IL-2- and IL-12-induced IFN- {gamma}
expression in murine NK cells. Blood. 2003 Mar 13 [Epub ahead of print]
IL-4/IL-5
• Enhanced mast cell histamine etc. secretion in response to IgE
• A Th2-like cytokine response is involved in bullous pemphigoid, the role of
IL-4 and IL-5 in the pathogenesis of the disease. Int J Immunopathol
Pharmacol. 1999 May-Aug;12(2):55-61.
IL-4/IL-6
IL-4/IL-10
IL-4/IL-11
• Synergistic interactions between interleukin- 1 1 and interleukin-4 in support
of proliferation of primitive hematopoietic progenitors of mice. Blood.
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1991 Sepl5;78(6):1448-51.
IL-4/IL-12
• Synergistic effects of IL-4 and IL- 1 8 on IL- 1 2-dependent IFN-garnma
production by dendritic cells. J Immunol 2000 Jan 1;164(1):64-71.
(increase Thl/Th2 differentiation)
• IL-4 synergistically enhances both IL-2- and IL-12-induced IFN- {gamma}
expression in murine NK cells. Blood. 2003 Mar 13 [Epub ahead of print]
IL-4/IL-13
• Abstract Interleukin-4 and interleukin-13 signaling connections maps.
Science. 2003 Jun 6;300(5625): 1527-8. (allergy, asthma)
• Inhibition of the IL-4/IL-13 receptor system prevents allergic sensitization
without affecting established allergy in a mouse model for allergic asthma.
J Allergy Clin Immunol. 2003 Jun;lll(6): 1361-1369.
IL-4/IL-16
• (asthma) Interleukin (IL)-4/IL-9 and exogenous IL-16 induce IL-16
production by BEAS-2B cells, a bronchial epithelial cell line. Cell
Immunol. 2001 Feb l;207(2):75-80
IL-4/IL-17
• Interleukin (IL)-4 and IL-17 synergistically stimulate IL-6 secretion in
human colonic myofibroblasts, hit J Mol Med. 2002 Nov;10(5):631-4.
(Gut inflammation)
IL-4/IL-24
• IL-24 is expressed by rat and human macrophages. Immunobiology. 2002
Jul;205(3):321-34.
IL-4/IL-25
• Abstract New IL-17 family members promote Thl or Th2 responses in the
lung: in vivo function of the novel cytokine IL-25. J Immunol. 2002 Jul
l;169(l):443-53. (allergic inflammation)
• Abstract Mast cells produce interleukin-25 upon Fcepsilon Rl-mediated
activation. Blood. 2003 May l;101(9):3594-6. Epub 2003 Jan 02. (allergic
inflammation)
IL-4/IFN-Y
• Abstract Interleukin 4 induces interleukin 6 production by endothelial cells:
synergy with interferon-gamma. Eur J Immunol. 1991 Jan;21 ( 1 ) : 97- 1 0 1 .
IL-4/SCF
• Regulation of human intestinal mast cells by stem cell factor and IL-4.
Immunol Rev. 2001 Feb; 179: 5 7-60. Review.
IL-5/IL-3
• Differential regulation of human eosinophil IL-3, IL-5, and GM-CSF
receptor alpha-chain expression by cytokines: IL-3, IL-5, and GM-CSF
down-regulate IL-5 receptor alpha expression with loss of IL-5
responsiveness, but up-regulate IL-3 receptor alpha expression. J Immunol.
2003 Jun 1;170(1 1):5359-66. (Allergic inflammation see abstract)
IL-5/IL-6
IL-5/IL-13
• Inhibition of allergic airways inflammation and airway
hyperresponsiveness in mice by dexamethasone: role of eosinophils, IL-5,
eotaxin, andIL-13. J Allergy Clin Immunol. 2003 May;ll 1(5): 1049-61.
IL-5/IL-17
• Interleukin- 17 orchestrates the granulocyte influx into airways after
allergen inhalation in a mouse model of allergic asthma. Am J Respir Cell
Mol Biol. 2003 Jan;28(l):42-50.
IL-5/IL-25
• Abstract New IL-17 family members promote Thl or Th2 responses in the
lung: in vivo function of the novel cytokine IL-25. J Immunol. 2002 Jul
l;169(l):443-53. (allergic inflammation)
• Abstract Mast cells produce interleukin-25 upon Fcepsilon Rl-mediated
activation. Blood. 2003 May l;101(9):3594-6. Epub 2003 Jan 02. (allergic
inflammation)
IL-5/IFN-y
IL-5/GM-CSF
• Differential regulation of human eosinophil IL-3, IL-5, and GM-CSF
receptor alpha-chain expression by cytokines: IL-3, IL-5, and GM-CSF
down-regulate IL-5 receptor alpha expression with loss of IL-5
responsiveness, but up-regulate IL-3 receptor alpha expression. J Immunol.
2003 Jun l;170(ll):5359-66. (Allergic inflammation)
IL-6/IL-10
IL-6/IL-11
IL-6/IL-16
• Interleukin- 16 stimulates the expression and production of pro-
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inflammatory cytokines by human monocytes. Immunology. 2000
May;100(l):63-9.
IL-6/IL-17
• Stimulation of airway mucin gene expression by interleukin (IL)-17
through IL-6 paracrine/autocrine loop. J Biol Chem. 2003 May
9;278(19): 17036-43. Epub 2003 Mar 06. (airway inflammation, asthma)
IL-6/IL-19
• Abstract IL-19 induces production of IL-6 and TNF-alpha and results in
cell apoptosis through TNF-alpha. J Immunol 2002 Oct 15;169(8):4288-
97.
IL-6/IFN-g
IL-7/IL-2
• Interleukin 7 worsens graft- versus-host disease. Blood. 2002 Oct
l;100(7):2642-9.
IL-7/IL-12
• Synergistic effects of IL-7 and IL-12 on human T cell activation. J
Immunol 1995 May 15;154(10):5093-102.
IL-7/IL-15
• Interleukin-7 and interleukin- 15 regulate the expression of the bcl-2 and c-
myb genes in cutaneous T-cell lymphoma cells. Blood. 2001 Nov
l;98(9):2778-83. (growth factor)
IL-8/IL-11
• Abnormal production of interleukin (IL)-1 1 and IL-8 in polycythaemia
vera. Cytokine. 2002 Nov 2 1;20(4): 178-83.
IL-8/IL-17
• The Role of IL-17 in Joint Destruction. Drug News Per sped. 2002
Jan;15(l): 17-23. (arthritis)
• Abstract Interleukin- 17 stimulates the expression of interleukin- 8, growth-
related oncogene-alpha, and granulocyte-colony-stimulating factor by
human airway epithelial cells. Am J Respir Cell Mo I Biol 2002
Jun;26(6):748-53. (airway inflammation)
IL-8/GSF
• Interleukin-8: an autocrine/paracrine growth factor for human
hematopoietic progenitors acting in synergy with colony stimulating factor-
1 to promote monocyte-macrophage growth and differentiation. Exp
Hematol. 1999 Jan;27(l):28-36.
IL-8/VGEF
• Intracavitary VEGF, bFGF, IL-8, IL-12 levels in primary and recurrent
malignant glioma. JNeurooncol 2003 May;62(3):297-303.
IL-9/IL-4
• Anti-interleukin-9 antibody treatment inhibits airway inflammation and
hyperreactivity in mouse asthma model. Am J Respir Crit Care Med. 2002
Aug 1;166(3):409-16.
IL-9/IL-5
• Pulmonary overexpression of IL-9 induces Th2 cytokine expression,
leading to immune pathology. J Clin Invest. 2002 Jan;109(l):29-39.
• Th2 cytokines and asthma. Interleukin-9 as a therapeutic target for asthma.
Respir Res. 2001;2(2):80-4. Epub 2001 Feb 15. Review.
• Abstract Interleukin-9 enhances interleukin-5 receptor expression,
differentiation, and survival of human eosinophils. Blood. 2000 Sep
15:96(61:2163-71 (asthma)
IL-9/IL-13
• Anti-interleukin-9 antibody treatment inhibits airway inflammation and
hyperreactivity in mouse asthma model. Am J Respir Crit Care Med. 2002
Aug 1;166(3):409-16.
• Direct effects of interleukin- 13 on epithelial cells cause airway
hyperreactivity and mucus overproduction in asthma. Nat Med. 2002
Aug;8(8):885-9.
IL-9/IL-16
• See IL-4/IL-16
IL-10/IL-2
• The interplay of interleukin- 1 0 (IL-1 0) and interleukm-2 (IL-2) in humoral
immune responses: IL-10 synergizes with IL-2 to enhance responses of
human B lymphocytes in a mechanism which is different from upregulation
of CD25 expression. Cell Immunol. 1994 Sep;157(2):478-88.
IL-10/IL-12
IL-10/TGF-P
• IL-10 and TGF-beta cooperate in the regulatory T cell response to mucosal
allergens in normal immunity and specific immunotherapy. Eur J
Immunol. 2003 May;33(5): 1205-14.
IL-10/IFN-y
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IL-ll/IL-6
• Interleukin-6 and interleukin-1 1 support human osteoclast formation by a
RANKL-independent mechanism. Bone, 2003 Jan;32(l):l-7. (bone
resorption in inflammation)
IL-ll/IL-17
• Polarized in vivo expression of IL-1 1 and IL-17 between acute and chronic
skin lesions. J Allergy Clin Immunol 2003 Apr;lll(4):875-81. (allergic
dermatitis)
• IL-17 promotes bone erosion in murine collagen-induced arthritis through
loss of the receptor activator of NF-kappa B ligand/osteoprotegerin
balance. J Immunol. 2003 Mar l;170(5):2655-62.
IL-11/TGF-P
• Polarized in vivo expression of IL-1 1 and IL-17 between acute and chronic
skin lesions. J Allergy Clin Immunol 2003 Apr;lll(4):875-81. (allergic
dermatitis)
IL-12/IL-13
• Relationship of Interleukin-1 2 and Interleukin-1 3 imbalance with class-
specific rheumatoid factors and anticardiolipin antibodies in systemic lupus
erythematosus. Clin Rheumatol 2003 May;22(2):107-ll.
IL-12/IL-17
• Upregulation of interleukin- 1 2 and - 1 7 in active inflammatory bowel
disease. Scand J Gastroenterol 2003 Feb;38(2): 180-5.
IL-12/IL-18
• Synergistic proliferation and activation of natural killer cells by interleukin
12 and interleukin 18. Cytokine. 1999 Nov;ll(l l):822-30.
• Inflammatory Liver Steatosis Caused by IL-12 and IL-18. J Interferon
Cytokine Res. 2003 Mar;23(3): 155-62.
IL-12/IL-23
• nterleukin-23 rather than interleukin- 12 is the critical cytokine for
autoimmune inflammation of the brain. Nature. 2003 Feb
13;421(6924):744-8.
• Abstract A unique role for IL-23 in promoting cellular immunity. J Leukoc
Biol 2003 Jan;73(l):49-56. Review.
IL-12/IL-27
• Abstract IL-27, a heterodimeric cytokine composed of EBB and p28
protein, induces proliferation of naive CD4(+) T cells. Immunity. 2002
Jun;16(6):779-90.
IL-12/IFN-y
• IL-12 induces IFN-y expression by B and T-cells as part of immune
stimulation.
IL-13/IL-5
• See IL-5/IL-13
IL-13/IL-25
• Abstract New IL-17 family members promote Thl or Th2 responses in the
lung: in vivo function of the novel cytokine IL-25. J Immunol 2002 Jul
l;169(l):443-53. (allergic inflammation)
• Abstract Mast cells produce interleukin-25 upon Fcepsilon Rl-mediated
activation. Blood. 2003 May l;101(9):3594-6. Epub 2003 Jan 02. (allergic
inflammation)
IL-15/IL-13
• Differential expression of interleukins (IL)-13 and IL-15 in ectopic and
eutopic endometrium of women with endometriosis and normal fertile
women. Am J Reprod Immunol 2003 Feb;49(2):75-83.
IL-15/IL-16
• IL-15 and IL-1 6 overexpression in cutaneous T-cell lymphomas: stage-
dependent increase in mycosis fungoides progression. Exp Dermatol 2000
Aug;9(4):248-51.
IL-15/IL-17
• Abstract IL-17, produced by lymphocytes and neutrophils, is necessary for
lipopolysaccharide-induced airway neutrophilia: IL-15 as a possible trigger.
J Immunol. 2003 Feb 15;170(4):2106-12. (airway inflammation)
IL-15/IL-21
• IL-2 1 m Synergy with IL-1 5 or IL-1 o Lnnances lr JN -gamma rroauction m
Human NK and T Cells. J Immunol 2003 Jun 1;170(1 1):5464-9.
IL-17/IL-23
• Interleukin-23 promotes a distinct CD4 T cell activation state characterized
by the production of interleukin- 17. J Biol Chem. 2003 Jan
17;278(3): 1910-4. Epub 2002 Nov 03
IL-17/TGF-P
• Polarized in vivo expression of IL-1 1 and IL-17 between acute and chronic
skin lesions. J Allergy Clin Immunol 2003 Apr;lll(4):875-81. (allergic
dermatitis)
IL-18/IL-12
• Svnergistic proliferation and activation of natural killer cells by interleukin
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12 and interleukin 18. Cytokine. 1999 Nov;ll(l l):822-30.
• Abstract Inhibition of in vitro immunoglobulin production by IL-12 in
murine chronic graft-vs.-host disease: synergism with IL-18. Eur J
Immunol. \yyo Jun ? Zo(oj.zui /-z*t.
IL-18/IL-21
• IL-2 1 in Synergy with IL-1 5 or IL-1 8 Enhances IFN-gamma Production in
Human NK and T Cells. J Immunol. 2003 Jun l;170(ll):5464-9.
IL-18/TGF-P
• Interleukin 18 and transforming growth factor betal in the serum of
patients with Graves' ophthalmopathy treated with corticosteroids. Int
Immunopharmacol. 2003 Apr;3(4): 549-52.
IL-18/IFN-Y
Anti-TNF
ALPHA/anti-CD4
• Synergistic therapeutic effect in DBA/1 arthritic mice.
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Annex 3: Oncology combinations
130
PCT/GB2003/002804
Target
Disease
Pair with
CD89*
Use as cytotoxic cell recruiter
all
CD19
B cell lymphomas
HLA-DR
CD5
HLA-DR
B cell lymphomas
CD89
CD19
CD5
CD38
Multiple myeloma
CD138
CDS 6
HLA-DR
CD138
Multiple myeloma
CD38
CD56
HLA-DR
CD138
Lung cancer
CD56
CEA
CD33
Acute myelod lymphoma
CD34
HLA-DR
CD56
Lung cancer
CD138
CEA
CEA
Pan carcinoma
MET receptor
VEGF
Pan carcinoma
MET receptor
VEGF
Pan carcinoma
MET receptor
receptor
IL-13
Asthma/pulmonary
IL-4
inflammation
IL-5
Eotaxin(s)
MDC
TARC
TNFoc
IL-9
EGFR
CD40L
IL-25
MCP-1
TGFP
EL-4
Asthma
IL-13
IL-5
Eotaxin(s)
MDC
TARC
TNFa
IL-9
EGFR
CD40L
IL-25
MCP-1
TGFp
Eotaxin
Asthma
IL-5
Eotaxin-2
Eotaxin-3
EGFR
cancer
HER2/neu
HER3
HER4
HER2
cancer
HER3
WO 2004/003019
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HER4
TNFR1
RA/Crohn's disease
IL-IR
IL-6R
IL-18R
A/OroTvn's disease
TT -loc/6
t..» ' J. UArf I-J
IL-6
IL-18
ICAM-1
IL-15
IL-17
IL-1R
RA/Crohn's disease
IL-6R
IL-18R
IL-18R
RA/Crohn's disease
IL-6R
WO 2004/003019
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PCT/GB2003/002804
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SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
PCT/GB2003/002804
134
Claims
1 . A dual-specific ligand comprising a first immunoglobulin single variable
domain having a binding specificity to a first epitope or antigen and a second
complementary immunoglobulin single variable domain having a binding activity
to a second epitope or antigen, wherein one or both of said antigens or epitopes
acts to increase the half-life of the ligand in vivo and wherein said first and second
domains lack mutually complementary domains which share the same specificity,
provided that said dual specific ligand does not consist of an anti-HS A V H domain
and an anti-p galactosidase V K domain.
2. A dual-specific ligand according to claim 1, comprising at least one single
heavy chain variable domain of an antibody and one complementary single light
chain variable domain of an antibody such that the two regions are capable of
associating to form a complementary VH/VL pair.
3 . A dual specific ligand according to claim 2 wherein the V H and V L are
provided by an antibody scFv fragment.
4. A dual-specific ligand according to claim 2 wherein the V H and V L are
provided by an antibody Fab region.
5. A four chain IgG immunoglobulin ligand comprising a dual specific
ligand of claim 2.
6. A four chain IgG immunoglobulin ligand according to claim 5, wherein
said IgG comprises two dual specific ligands, said dual specific ligands being
identical in their variable domains.
7. A four chain IgG immunoglobulin ligand according to claim 5, wherein
said IgG comprises two dual specific ligands, said dual specific ligands being
different in their variable domains.
8. A ligand comprising a first immunoglobulin variable domain having a first
antigen or epitope binding specificity and a second immunoglobulin variable
domain having a second antigen or epitope binding specificity wherein one or
both of said first and second variable domains bind to an antigen which increases
the half-life of the ligand in vivo, and the variable domains are not complementary
to one another.
9. A ligand according to claim 8 wherein the first and the second
immunoglobulin variable domains are heavy chain variable domains (V H ).
10. A ligand according to claim 8 wherein the first and the second
immunoglobulin variable domains are light chain variable domains (V L ).
11. A ligand according to claim any preceding claim, wherein the first and
second epitopes bind independently, such that the dual specific ligand may
simultaneously bind both the first and second epitopes or antigens.
SU^fif iSWifi^^UlL^ 26)
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12. A ligand according to claim 11, wherein the dual specific ligand comprises
a first form and a second form in equilibrium in solution, wherein both epitopes or
antigens bind to the first form independently but compete for binding to the
second form.
13. A ligand according to any preceding claim wherein the variable regions
are derived from immunoglobulins directed against said epitopes or antigens.
14. A ligand according to any preceding claim, wherein said first and second
epitopes are present on separate antigens.
15. A ligand according to any one of claims 1 to 1 1, wherein said first and
second epitopes are present on the same antigen.
16. A ligand according to any preceding claim comprising a variable domain
that is derived from a repertoire of single antibody domains.
17. A ligand of claim 16 wherein said repertoire is displayed on the surface of
filamentous bacteriophage and wherein the single antibody domains are selected
by binding of the bacteriophage repertoire to antigen.
18. A ligand according to any preceding claim wherein the sequence of at
least one variable domain is modified by mutation or DNA shuffling.
19. A dual-specific ligand according to any preceding claim wherein the
variable regions are non-covalently associated.
20. A dual-specific ligand according to any one of claims 1 to 1 8 wherein the
variable regions are covalently associated.
21 . A dual-specific ligand according to claim 20 wherein the covalent
association is mediated by disulphide bonds.
22. A dAb monomer ligand specific for TNFa, which dissociates from human
TNFa with a dissociation constant (K d ) of 50nM to 20pM, and a K Q ff rate constant
of 5xl0 _1 to lxlO" 7 s" 1 , as determined by surface plasmon resonance.
23. A dAb monomer ligand specific for TNFa according to claim 22, wherein
the dAb is a Vk.
24. A dAb monomer ligand specific for TNF receptor 1 (p55), which
dissociates from human TNF receptor 1 with a dissociation constant (K d ) of 50nM
to 20pM, and aKoff rate constant of 5x1 0" 1 to lxl 0" 7 s" 1 , as determined by surface
plasmon resonance.
25. A dAb monomer ligand according to claim 22 to claim 24, wherein the
monomer neutralises human TNFa or TNF receptor 1 in a standard cell assay
with anND50 of 500nM to 50pM.
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26. A dAb monomer ligand specific for TNF receptor 1 (p55), wherein the
dAb antagonises the activity of the TNF receptor 1 in a standard cell assay with
an ND 50 of <100nM, and at a concentration of <10pM the dAb agonises the
activity of the TNF receptor 1 by <5% in the assay.
27. A dAb monomer ligand specific for serum albumin (SA) which dissociates
from SA with a dissociation constant (K d ) of InM to SOO^M, as determined by
surface plasmon resonance.
28. A dAb monomer ligand according to claim 27, wherein the monomer
binds SA in a standard ligand binding assay with an IC50 of InM to 500jaM.
29. A dAb monomer ligand specific for TNFoc, wherein the dAb comprises
the amino acid sequence of TAR1-5-19 or a sequence that is at least 80%
homologous thereto.
30. A dAb monomer ligand specific for TNFoc, wherein the dAb comprises
the amino acid sequence of TAR1-5 or a sequence that is at least 80%
homologous thereto.
31. A dAb monomer ligand specific for TNFa, wherein the dAb comprises
the amino acid sequence of TAR1-27 or a sequence that is at least 80%
homologous thereto.
32. A dAb monomer ligand specific for TNF receptor 1, wherein the dAb
comprises the amino acid sequence of TAR2-10 or a sequence that is at least 80%
homologous thereto.
33. A dAb monomer ligand specific for TNF receptor 1, wherein the dAb
comprises the amino acid sequence of TAR2-10 or a sequence that is at least 90%
homologous thereto.
34. A dAb monomer ligand specific for TNF receptor 1 , wherein the dAb
comprises the amino acid sequence of TAR2-5 or a sequence that is at least 80%
homologous thereto.
35. A dAb monomer ligand specific for TNF receptor 1 , wherein the dAb
comprises the amino acid sequence of TAR2-5 or a sequence that is at least 90%
homologous thereto.
36. A dAb monomer ligand specific for SA, wherein the dAb comprises the
amino acid sequence of MSA- 16 or a sequence that is at least 80% homologous
thereto.
37. A dAb monomer ligand specific for SA, wherein the dAb comprises the
amino acid sequence of MSA-26 or a sequence that is at least 80% homologous
thereto.
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38. A dAb monomer according to any one of claims 29 to 37, wherein the
TNFa, TNF receptor 1 or S A is in human form.
39. A dAb monomer further comprising a terminal Cys residue.
40. A dAb monomer according to any one of claims 29 to 38 5 further
comprising a terminal Cys residue.
41. A dual specific ligand comprising at least one dAb monomer according to
any one of claims 22 to 40.
42. A dual specific ligand according to claim 41, which is a dimer.
43 . A dual specific ligand according to claim 42, wherein the dimer comprises
anti-human TNF alpha dAb according to any one of claims 22, 23 and 28-30, and
an anti-SA dAb according to any one of claims 26, 27 and 34-36.
44. A dual specific ligand according to claim 42, wherein the dimer is a
homo- or hetero-dimer comprising first and second anti-human TNF alpha dAbs,
each dAb being according to any one of claims 22, 23 and 28-30.
45. A dual specific ligand according to claim 41, which is a trimer.
46. A dual specific ligand according to claim 45, which is a homotrimer
comprising three copies of an anti-human TNF alpha dAb according to any one of
claims 22, 23 and 29-3 1 .
47. A ligand according to any preceding claim, which comprises a universal
framework.
48. A ligand according to claim 47, wherein the universal framework
comprises a Vh framework selected from the group consisting of DP47, DP45 and
DPS 8; and/or the V L framework is DPK9.
49. A ligand according to any preceding claim which comprises a binding site
for a generic ligand.
50. The ligand of claim 49, wherein the generic ligand binding site is selected
from the group consisting of protein A, protein L and protein G.
51. A ligand according to any preceding claim, wherein the ligand comprises a
variable domain having one or more framework regions comprising an amino acid
sequence that is the same as the amino acid sequence of a corresponding
framework region encoded by a human germline antibody gene segment, or the
amino acid sequences of one or more of said framework regions collectively
comprises up to 5 amino acid differences relative to the amino acid sequence of
said corresponding framework region encoded by a human germline antibody
gene segment.
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52. A ligand according to any one of claims 1 to 51 5 wherein the ligand
comprises a variable domain, wherein the amino acid sequences of FW1, FW2,
FW3 and FW4 are the same as the amino acid sequences of corresponding
framework regions encoded by a human germline antibody gene segment, or the
amino acid sequences of FW1, FW2, FW3 and FW4 collectively contain up to 10
amino acid differences relative to the amino acid sequences of corresponding
framework regions encoded by said human germline antibody gene segment.
53. The ligand according to claim 51 or claim 52, which comprises an
antibody variable domain comprising FW1, FW2 and FW3 regions, and the
amino acid sequence of said FW1, FW2 and FW3 are the same as the amino acid
sequences of corresponding framework regions encoded by human germline
antibody gene segments.
54. The ligand according to any one of claims 51 to 53, wherein said human
germline antibody gene segment is selected from the group consisting of DP47,
DP45, DP48 and DPK9.
55. A ligand according to any preceding claim, comprising a V H domain that
is not a Camelid immunoglobulin variable domain.
56. The ligand of Claim 55, comprising a V H domain that does not contain one
or more amino acids that are specific to Camelid immunoglobulin variable
domains as compared to human Vh domains.
57. A method for producing a ligand comprising a first immunoglobulin single
variable domain having a first binding specificity and a second single
immunoglobulin single variable domain having a second binding specificity, one
or both of the binding specificities being specific for a protein which increases the
half-life of the ligand in vivo, the method comprising the steps of:
(a) selecting a first variable domain by its ability to bind to a first epitope,
(b) selecting a second variable region by its ability to bind to a second
epitope,
(c) combining the variable regions; and
(d) selecting the ligand by its ability to bind to said first and second epitopes;
wherein, when said variable domains are complementary, neither of said domains
is a Vh domain specific for HSA.
58. A method according to claim 57 wherein said first variable domain is
selected for binding to said first epitope in absence of a complementary variable
domain.
59. A method according to claim 57 wherein said first variable domain is
selected for binding to said first epitope in the presence of a third complementary
variable domain in which said third variable domain is different from said second
variable domain.
60. Nucleic acid encoding a dual-specific ligand according to any one of
claims 1 to 56.
SUBSTITUTE SHEET (RULE 26)
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61 . A nucleic acid according to claim 60 which is specific for TNFoc,
comprising the nucleic acid sequence of TAR1-5-19 or a sequence that is at least
70% homologous thereto.
62. A nucleic acid according to claim 60 which is specific for TNFa,
comprising the nucleic acid sequence of TAR1-5 or a sequence that is at least
70% homologous thereto.
63 . A nucleic acid according to claim 60 which is specific for TNFa,
comprising the nucleic acid sequence of TAR 1-27 or a sequence that is at least
70% homologous thereto.
64. A nucleic acid according to claim 60 which is specific for TNF receptor 1,
comprising the nucleic acid sequence of TAR2-10 or a sequence that is at least
70% homologous thereto.
65. A nucleic acid according to claim 60 which is specific for TNF receptor 1 5
comprising the nucleic acid sequence of TAR2-10 or a sequence that is at least
80% homologous thereto.
66. A nucleic acid according to claim 60 which is specific for TNF receptor 1 ,
comprising the nucleic acid sequence of TAR2h-5 or a sequence that is at least
70% homologous thereto.
67. A nucleic acid according to claim 60 which is specific for TNF receptor 1,
comprising the nucleic acid sequence of TAR2h-5 or a sequence that is at least
80% homologous thereto.
68. A nucleic acid according to claim 60 which is specific for SA, comprising
the nucleic acid sequence of MSA- 16 or a sequence that is at least 70%
homologous thereto.
69. A nucleic acid according to claim 60 which is specific for SA, comprising
the nucleic acid sequence of MSA-26 or a sequence that is at least 70%
homologous thereto.
70. A vector comprising nucleic acid according to any one of claims 60 to 69.
71 . A vector according to claim 70, further comprising components necessary
for the expression of a dual-specific ligand.
72. A host cell transfected with a vector according to claim 71 .
73 . A method for producing a closed conformation multi-specific ligand
comprising a first single epitope binding domain having a first epitope binding
specificity and a non-complementary second epitope binding domain having a
second epitope binding specificity, wherein the first and second binding
specificities are capable of competing for epitope binding such that the closed
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conformation multi-specific ligand may not bind both epitopes simultaneously,
said method comprising the steps of:
a) selecting a first epitope binding domain by its ability to bind to a first
epitope,
b) selecting a second epitope binding domain by its ability to bind to a
second epitope,
c) combining the epitope binding domains such that the domains are in a
closed conformation; and
d) selecting the closed conformation multispecific ligand by its ability to bind
to said first second epitope and said second epitope, but not to both said first and
second epitopes simultaneously.
74. A method according to claim 73 wherein the first and the second epitope
binding domains are immunoglobulin variable heavy chain domains (Vh)*
75. A method according to claim 73 wherein the first and the second
immunoglobulin variable domains are immunoglobulin variable light chain
domains (Vl)-
76. A method according to any one of claims 73 to 75 wherein the
immunoglobulin domains are derived from immunoglobulins directed against said
epitopes.
77. A method according to any one of claims 73 to 76, wherein said first and
second epitopes are present on separate antigens.
78. A method according to any one of claims 73 to 76, wherein said first and
second epitopes are present on the same antigen.
79. A method according to any one of claims 73 to 78 wherein the variable
domain is derived from a repertoire of single antibody domains.
80. A method of claim 79 wherein said repertoire is displayed on the surface
of filamentous bacteriophage and wherein the single antibody domains are
selected by binding of the bacteriophage repertoire to antigen.
81 . A method of any one of claims 73 to 80 wherein the sequence of at least
one immunoglobulin variable domain is modified by mutation or DNA shuffling.
82. A closed conformation multispecific ligand comprising a first epitope
binding domain having a first epitope binding specificity and a non-
complementary second epitope binding domain having a second epitope binding
specificity wherein the first and second binding specificities are capable of
competing for epitope binding such that the closed conformation multi-specific
ligand cannot bind both epitopes simultaneously.
83. A closed conformation multispecific ligand according to claim 82,
obtainable by a method according to any one of claims 73 to 80.
SUBSTITUTE SHEET (RULE 26)
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84. A closed conformation multispecific ligand according to claim 82 or claim
83, comprising more than one single heavy chain variable domain of an antibody
or more than one light chain variable domain of an antibody.
85. A closed conformation multi-specific ligand according to claim 84
wherein the Vh an d Vl are linked by a peptide linker.
86. A closed conformation multi-specific ligand according to claim 84
wherein the Vh or Vl are provided by an antibody Fab-like region.
87. A closed conformation multi-specific ligand according to any one of
claims 82 to 84 wherein the variable regions are non-covalently associated.
88. A closed conformation multi-specific ligand according to any one of
claims 82 to 84 wherein the variable regions are covalently associated.
89. A closed conformation multi-specific ligand according to claim 87
wherein the covalent association is mediated by disulphide bonds.
90. A closed conformation multi-specific ligand according to any of claims 82
to 89 which comprises a universal framework.
91. A closed conformation multi-specific ligand according to any of claims 82
to 90 which comprises a binding site for a generic ligand.
92. The closed conformation multi-specific ligand of claim 91 , wherein the
generic ligand binding site is selected from the group consisting of protein A,
protein L and protein G.
93. A closed conformation ligand according to any of claims 82 to 92,
wherein the ligand comprises a variable domain having one or more framework
regions comprising an amino acid sequence that is the same as the amino acid
sequence of a corresponding framework region encoded by a human germline
antibody gene segment, or the amino acid sequences of one or more of said
framework regions collectively comprises up to 5 amino acid differences relative
to the amino acid sequence of said corresponding framework region encoded by a
human germline antibody gene segment.
94. The closed conformation ligand according to claim 93, wherein the ligand
comprises a variable domain wherein the amino acid sequences of FW1, FW2,
FW3 and FW4 are the same as the amino acid sequences of corresponding
framework regions encoded by a human germline antibody gene segment, or the
amino acid sequences of FW1, FW2, FW3 and FW4 collectively contain up to 10
amino acid differences relative to the amino acid sequences of corresponding
framework regions encoded by said human germline antibody gene segment.
95. The closed conformation ligand according to claim 93 or claim 94, which
comprises an antibody variable domain comprising FW1, FW2 and FW3 regions,
SUBSTITUTE SHEET (RULE 26)
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and the amino acid sequence of said FW1, FW2 and FW3 are the same as the
amino acid sequences of corresponding framework regions encoded by human
germline antibody gene segments.
96. The closed conformation ligand according to any one of claims 92 to 95,
wherein said human germline antibody gene segment is selected from the group
consisting of DP47, DP45, DP48 and DPK9.
97. A closed conformation ligand according to any one of claims 92 to 96,
comprising a Vh domain that is not a Camelid immunoglobulin variable domain.
98. The closed conformation ligand of Claim 97 ? wherein the V H domain does
not contain one or more amino acids that are specific to Camelid immunoglobulin
variable domains as compared to human Vh domains.
99. A closed conformation multi-specific ligand according to any one of
claims 82 to 98, wherein one specificity thereof is for an agent effective to
increase the half life of the ligand.
100. A kit comprising a closed conformation multi-specific ligand according to
any one of claims 82 to 99.
101. Nucleic acid encoding at least a closed conformation multispecific ligand
according to any one of claims 82 to 99.
1 02. A vector comprising nucleic acid according to claim 101.
103. A vector according to claim 102, further comprising components
necessary for the expression of a closed conformation multispecific ligand.
104. A host cell transfected with a vector according to claim 103.
105. A method for detecting the presence of a target molecule, comprising:
(a) providing a closed conformation multispecific ligand bound to an agent, said
ligand being specific for the target molecule and the agent, wherein the agent
which is bound by the ligand leads to the generation of a detectable signal on
displacement from the ligand;
(b) exposing the closed conformation multispecific ligand to the target molecule;
and
(c) detecting the signal generated as a result of the displacement of the agent.
106. A method according to claim 105, wherein the agent is an enzyme, which
is inactive when bound by the closed conformation multispecific ligand.
107. A method according to claim 105, wherein the agent is the substrate for an
enzyme
108. A method according to claim 107, wherein the agent is a fluorescent,
luminescent or chromogenic molecule which is inactive or quenched when bound
by the ligand.
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109. A kit for performing a method according to any one of claims 105-108,
comprising a closed conformation multispecific ligand capable of binding to a
target molecule, and optionally an agent and buffers suitable therefor.
110. A homogenous immunoassay incorporating a method according to any
one of claims 105-108.
111. A ligand according to any one of claims 1 to 56 for use in therapy.
112. A pharmaceutical composition comprising a ligand according to any one
of claims 1 to 56 ? and a pharmaceutical^ acceptable eccipient, carrier or diluent.
113. A method for preparing a chelating multimeric ligand comprising the steps
of:
(a) providing a vector comprising a nucleic acid sequence encoding a
single binding domain specific for a first epitope on a target;
(b) providing a vector encoding a repertoire comprising second binding
domains specific for a second epitope on said target, which epitope can be the
same or different to the first epitope, said second epitope being adjacent to said
first epitope; and
(c) expressing said first and second binding domains; and
(d) isolating those combinations of first and second binding domains which
combine together to produce a target-binding dimer.
114. A method according to claim 113, wherein the first and second binding
domains are associated covalently through a linker.
115. A method according to claim 113, wherein the first and second binding
domains are associated non-covalently.
116. A method according to claim 113, wherein the first and second binding
domains are associated through natural association of the domains.
117. A method according to claim 116, wherein the binding domains comprise
a Vh domain and a V K domain.
SUBSTITUTE SHEET (RULE 26)
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WO 2004/003019
PCT/GB2003/002804
2/17
FIG. 2
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r
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promoter
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linker peptide
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plT1/plT2
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amp
gm
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RBS
CAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGCAAATTCTATTTCAAGGAGACAGTCATA ATG AAA TAC CTA
— > * M K Y L
LMB3
Sf il
Ncol
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LPTAAAGLLLLAAQPAMAEVF
Xhol linker
GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GT C TCG AG C GGT GGA GGC GGT TCA GGC GGA GGT
DYWGQGTLVTVS SGGGGSGGG
Sail NotI
GGC AGC GGC GGT GGC GG G TCG AC G GAC ATC CAG ATG ACC CAG GCG GCC GCA GAA CAA AAA CTC
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CAT CAT CAT CAC CAT CAC GGG GCC GCA
HHHHHHGAA
(insertion in pIT2 only)
my c- tag Gene III
ATC TCA GAA GAG GAT CTG AAT GGG GCC GCA TAG ACT GTT GAA AGT TGT TTA GCA AAA CCT CAT
ISEEDLNGAA* TVESCLAKPH
< ■
pHEN seq
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
PCT/GB2003/002804
3/17
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SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
PCT/GB2003/002804
4/17
Q
o
1.6
1.4
1.2
1
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FIG. 4
Phage ELISA of a dual specific ScFv antibody K8
[Mliil
3 4 5
Antigens
1- HSA
2- APS
3- b-gal
4- Peanut
5- BSA
6- iysosyme
7- cytochrome c
FIG. 5
Soluble ELISA of the Dual Specific ScFv Antibody K8
2i
600
K8 ScFv concentration (nmol)
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
PCT/GB2003/002804
5/17
o
O
1.6
1.4-
1.2-
1 -
0.8
0.6
0.4-
0.2-
0
FIG. 6
Soluble ScFv ELISA of K8V /dummy V H clone
1
2 3
Antigens
1- BSA
2- b-gal
3- APS
4- Protein A
FIG. 7
RBS
CAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGCAAATTCTATTTCAAGGAGACAGTCATA ATG AAA TAC CTA
- „> M K Y L
LMB3
Sfil Ncol
TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GC G GCC CAG CCG GCC ATG GCC GAG GTG TTT
L P T AAAGIiL L LAA Q P ~ A M A E V F
>
Xhol linker
GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GT C TCG AG C GGT GGA GGC GGT TCA GGC GGA GGT
DYWGQGTLVTVS SGGGGSGGG
Sail NotI
GGC AGC GGC GGT GGC GG G TCG AC G GAC ATC CAG ATG ACC CAG GCG GCC GC A GAA CAA AAA CTC "
GSGGGGSTT) IQMTQA ~ A " A £7 Q K L
<
link seg new
HIS -tag
CAT CAT CAT CAC CAT CAC GGG GCC GCA
HHHHHHGAA
(insertion in V domain vector 2 only
myc-tag Gene III
ATC TCA GAA GAG GAT CTG AAT GGG GCC GCA TAG ACT GTT GAA AGT TGT TTA GCA AAA CCT CAT
ISEEDLNGAA*TVESCLAKPH
<
pHEN seq
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
PCT/GB2003/002804
6/17
lac RBS
promoter
-^p15 or\y
FIG. 8
o
o
Is
leader linker
CH vector
o
CO
{M13ori>
^ — cc
CH gene myc 2x
tag ochre
Cm
FIG. 9
o
CO CO
— II — MB
lac
promoter
RBS leader
CK vector
-(colEI orj)-
amp
• * • ■ » • »
CK gene
CD
O
CO
o
flag
tag
S3
2x
ochre
<M13ori>
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
PCT/GB2003/002804
7/17
^ 160
o
o
LL.
FIG. 10
TNF receptor assay
0.1 1 10 100 1000
dAb/dimer concentration nM
PEP1-5d4
PEP1-5-19d4
PEP1-519 monomer
FIG. 11
o
o
CO
LL
z:
TNF receptor assay
1 10 100
dimer concentration nM
1000
-x-
PEP1-5d1
PEP1-5d2
PEP1-5d3
PEP1-5d4
PEP1-5d5
PEP1-5d6
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
8/17
PCT/GB2003/002804
FIG. 12
TNF Receptor assay
110
-10-J 1
0.1 1 10 100 1000
dAb/dimer concentration nM
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
9/17
PCT/GB2003/002804
FIG. 13
Dummy V H sequence for library 1
1
E
GAG
CTC
V
GTG
CAC
Q
CAG
GTC
L
CTG
GAC
L
TTG
AAC
E
GAG
CTC
S
TCT
AGA
G
GGG
CCC
G
GGA
CCT
G
GGC
CCG
L
TTG
AAC
V
GTA
CAT
Q
CAG
GTC
P
CCT
GGA
G
GGG
CCC
G
GGG
CCC
49
S
TCC
AGG
L
CTG
GAC
R
CGT
GCA
T i
CTC
GAG
S
TCC
AGG
C
TGT
ACA
A
GCA
CGT
A
GCC
CGG
S
TCC
AGG
G
GGA
CCT
F
TTC
AAG
T
ACC
TGG
F
TTT
AAA
S
AGC
TCG
S
AGC
TCG
Y
TAT
ATA
97
A
GCC
CGG
M
ATG
TAC
S
AGC
TCG
W
TGG
ACC
V
GTC
CAG
R
CGC
GCG
Q
CAG
GTC
A
GCT
CGA
P
CCA
GGT
G
GGG
CCC
K
AAG
TTC
G
GGT
CCA
L
CTA
GAT
E
GAG
CTC
W
TGG
ACC
V
GTC
CAG
S
A
I
S
G
S
G
G
S
T
Y
Y
A
D
S
V
145
TCA
GCT
ATT
AGT
GGT
AGT
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ACA
TAC
TAC
GCA
GAC
TCC
GTG
AGT
CGA
TAA
TCA
CCA
TCA
CCA
CCA
TCG
TGT
ATG
ATG
CGT
CTG
AGG
CAC
193
K
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TTC
G
GGC
CCG
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CGG
GCC
F
TTC
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T
ACC
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I
ATC
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TCC
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R
CGT
GCA
D
GAC
CTG
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AAT
TTA
S
TCC
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K
AAG
TTC
N
AAC
TTG
T
ACG
TGC
L
CTG
GAC
Y
TAT
ATA
241
L
CTG
GAC
Q
CAA
GTT
M
ATG
TAC
N
AAC
TTG
S
AGC
TCG
L
CTG
GAC
R
CGT
GCA
A
GCC
CGG
E
GAG
CTC
D
GAC
CTG
T
ACC
TGG
A
GCG
CGC
V
GTA
CAT
Y
TAT
ATA
Y
TAC
ATG
C
TGT
ACA
A
K
S
Y
G
A
F
D
Y
W
G
Q
G
T
L
V
289
GCG
AAA
AGT
TAT
GGT
GCT
TTT
GAC
TAC
TGG
GGC
CAG
GGA
ACC
CTG
GTC
CGC
TTT
TCA
ATA
CCA
CGA
AAA
CTG
ATG
ACC
CCG
GTC
CCT
TGG
GAC
CAG
337
T
ACC
TGG
V
GTC
CAG
S
TCG
AGC
S
AGC
TCG
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
10/17
PCT/GB2003/002804
FIG. 14
Dummy V H sequence for library 2
1
1
E
GAG
CTC
V
GTG
CAC
Q
CAG
GTC
L
CTG
GAC
L
TTG
AAC
E
GAG
CTC
S
TCT
AGA
G
GGG
CCC
G
GGA
CCT
G
GGC
CCG
L
TTG
AAC
V
GTA
CAT
Q
CAG
GTC
P
CCT
GGA
G
GGG
CCC
G
GGG
CCC
49
S
TCC
AGG
L
CTG
GAC
R
CGT
GCA
L
CTC
GAG
S
TCC
AGG
C
TGT
ACA
A
GCA
CGT
A
GCC
CGG
S
TCC
AGG
G
GGA
CCT
F
TTC
AAG
T
ACC
TGG
F
TTT
AAA
S
AGC
TCG
S
AGC
TCG
Y
TAT
ATA
97
A
GCC
CGG
M
ATG
TAC
S
AGC
TCG
W
TGG
ACC
V
GTC
CAG
R
CGC
GCG
Q
CAG
GTC
A
GCT
CGA
P .
CCA
GGT
G
GGG
CCC
K
AAG
TTC
G
GGT
CCA
T i
CTA
GAT
E
GAG
CTC
W
TGG
ACC
V
GTC
CAG
S
A
I
S
G
S
G
G
S
T
Y
Y
A
D
S
V
145
TCA
GCT
ATT
AGT
GGT
AGT
GGT
GGT
AGC
ACA
TAC
TAC
GCA
GAC
TCC
GTG
AGT
CGA
TAA
TCA
CCA
TCA
CCA
CCA
TCG
TGT
ATG
ATG
CGT
CTG
AGG
CAC
193
K
AAG
TTC
G
GGC
CCG
R
CGG
GCC
F
TTC
AAG
T
ACC
TGG
I
ATC
TAG
S
TCC
AGG
R
CGT
GCA
D
GAC
CTG
N
AAT
TTA
S
TCC
AGG
K
AAG
TTC
N
AAC
TTG
T
ACG
TGC
L
CTG
GAC
Y
TAT
ATA
241
L
CTG
GAC
Q
CAA
GTT
M
ATG
TAC
N
AAC
TTG
S
AGC
TCG
L
CTG
GAC
R
CGT
GCA
A
GCC
CGG
E
GAG
CTC
D
GAC
CTG
T
ACC
TGG
A
GCG
CGC
V
GTA
CAT
Y
TAT
ATA
Y
TAC
ATG
C
TGT
ACA
A
K
S
Y
G
A
X
X
X
X
F
D
Y
W
G
Q
289
GCG
AAA
AGT
TAT
GGT
GCT
NNK
NNK
NNK
NNK
TTT
GAC
TAC
TGG
GGC
CAG
CGC
TTT
TCA
ATA
CCA
CGA
NNK
NNK
NNK
NNK
AAA
CTG
ATG
ACC
CCG
GTC
337
G
GGA
CCT
T
ACC
TGG
L
CTG
GAC
V
GTC
CAG
T
ACC
TGG
V
GTC
CAG
S
TCG
AGC
S
AGC
TCG
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
11 I M
PCT/GB2003/002804
FIG. 15
Dummy V K sequence for library 3
DIQMTQSPSSLSASVG
1 GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA
CTG TAG GTC TAC TGG GTC AGA GGT AGG AGG GAC AGA CGT AGA CAT CCT
DRVTITCRASQS I S S Y
4 9 GAC CGT GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC ATT AGC AGC TAT
CTG GCA CAG TGG TAG TGA ACG GCC CGT TCA GTC TCG TAA TCG TCG ATA
LNWYQQKPGKAPKLL I
97 TT A AAT TGG TAC CAG CAG AAA CCA GGG AAA GCC CCT AAG CTC CTG ATC
AAT TTA ACC ATG GTC GTC TTT GGT CCC TTT CGG GGA TTC GAG GAC TAG
Y_AASSLQSGVPSRFSG
14 5 TA T GCT GCA TCC AGT TTG CAA AGT GGG GTC CCA TCA CGT TTC AGT GGC
ATA CGA CGT AGG TCA AAC GTT TCA CCC CAG GGT AGT GCA AAG TCA CCG
SGSGTDFTLTISSLQP
193 AGT GGA TCT GGG ACA GAT TTC ACT CTC ACC ATC AGC AGT CTG CAA CCT
TCA CCT AGA CCC TGT CTA AAG TGA GAG TGG TAG TCG TCA GAC GTT GGA
EDFATYYCQQ S Y S T P N
2 41 GAA GAT TTT GCT ACG TAC TAC TGT CAA CAG AGT TAC AGT ACC CCT AAT
CTT CTA AAA CGA TGC ATG ATG ACA GTT GTC TCA ATG TCA TGG GGA TTA
TFGQGTKVEIKR
2 89 ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA CGG
TGC AAG CCG GTT CCC TGG TTC CAC CTT TAG TTT GCC
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
PCT/GB2003/002804
12/17
FIG. 16
Nucleotide and amino acid sequence of anti MSA dAbs MSA 16 and MSA 26
A: MSA 16
GAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT
DIQMTQSPSSLSAS
GTA GGA GAC CGT GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC
VGDRVTITCRASQS
ATT ATT AAG CAT TTA AAG TGG TAC CAG CAG AAA CCA GGG AAA
1 I K H L K W ... YQQKPGK
GCC CCT AAG CTC CTG ATC TAT GGT GCA TCC CGG TTG CAA AGT
APKLLIYGASRLQS
GGG GTC CCA TCA CGT TTC AGT GGC AGT GGA TCT GGG ACA GAT
GVPSRFSGSGSGTD
TTC ACT CTC ACC ATC AGC AGT CTG CAA CCT GAA GAT TTT GCT
FTLTISSLQPEDFA
ACG TAC TAC TGT CAA CAG GGG GCT CGG TGG CCT CAG ACG TTC
TYYCQQGARWPQ TF
GGC CAA GGG ACC AAG GTG GAA ATC AAA CGG
GQGTKVEIKR
B: MSA 26
GAC ATC CAG ATG ACC CAG TCT
D I Q M T Q S
GTA GGA GAC CGT GTC ACC ATC
V G D R V T I
ATT TAT TAT CAT TTA AAG TGG
I Y Y H L K W
GCC CCT AAG CTC CTG ATC TAT
A P K L L I Y
GGG GTC CCA TCA CGT TTC AGT
G V P S R F S
TTC ACT CTC ACC ATC AGC AGT
F T L T I S S
ACG TAC TAC TGT CAA CAG GTT
T Y Y C Q Q V
GGC CAA GGG ACC AAG GTG GAA
G Q G T K V E
CCA TCC TCC CTG TCT GCA TCT
P S S L S A S
ACT TGC CGG GCA AGT CAG AGC
T C R A S Q S
TAC CAG CAG AAA CCA GGG AAA
YQQKPGK
AAG GCA TCC ACG TTG CAA AGT
K A S T L Q S
GGC AGT GGA TCT GGG ACA GAT
G S G S G T D
CTG CAA CCT GAA GAT TTT GCT
L Q P E D F A
CGG AAG GTG CCT CGG ACG TTC
R K V P R T F
ATC AAA CGG
I K R
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
PCT/GB2003/002804
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
PCT/GB2003/002804
14/17
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019 PCT/GB2003/002804
15/17
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
PCT/GB2003/002804
16/17
(a)
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
FIG. 19
m Series 1
TAR 1-5- TAR 1-5-
19dAb 19dAb
+ MSA
Dual Dual
specific specific
+ MSA
(b)
biotinylated anti-TNF
TNF
chromogenic
substrate
Streptavidin-HRP
TNFRI/Fc chimera
Anti-Fc capturing antibody
(c)
100
~ 80
o
o
CO
LL
60
40
5 20
0
-20
't*t*t i * *lal
LH
TNF Receptor assay
LH +
MSA
1 nnnnnnnno"
TNF +
MSA
MSA
only
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
17/17
PCT/GB2003/002804
FIG. 20
TNF Receptor assay
120 n
100
o
o
CD
• HAS
_Q
Ll_
80
60
18nM LH + MSA
MSA
18nM LH + HSA
HSA
Serum Albumin (mg/ml)
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
1
PCT/GB2003/002804
CD
CO
LU
U
LU
C?
LU
CO
Eh
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CD
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CD
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SUBSTITUTE SHEET (RULE 26)
WO 2004/003019 PCT/GB2003/002804
13
cd cj
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SUBSTITUTE SHEET (RULE 26)
(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property
Organization
International Bureau
(43) International Publication Date
8 January 2004 (08.01.2004)
PCT
(10) International Publication Number
WO 2004/003019 A3
(51) International Patent Classification 7 : C07K 16/24,
16/18, 16/28, A61K 39/395, C07K 16/46, C12N 15/63,
15/62, 15/13
(21) International Application Number:
PCT/GB2003/002804
(22) International Filing Date: 30 June 2003 (30.06.2003)
(25) Filing Language: English
(74) Agents: MASCHIO, Antonio et aL; D Young & Co, 21
New Fetter Lane, London EC4A IDA (GB).
(81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
CZ, DE, DK, DM, DZ, EC, EE, ES, K, GB, GD, GE, GH,
GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
MX, MZ, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RU, SC,
SD, SE, SG, SK, SL, SY, TJ, TM, TN, TR, TT, TZ, UA,
UG, US, UZ, VC, VN, YU, ZA, ZM, ZW.
(26) Publication Language:
English
(30) Priority Data:
PCT/GB02/03014 28 June 2002 (28.06.2002) GB
0230202.4 27 December 2002 (27.12.2002) GB
(71) Applicant (for all designated States except US): DOMAN-
TIS LIMITED [GB/GB]; 315 Cambridge Science Park,
Cambridge, Cambridgeshire CB4 0WG (GB).
(72) Inventors; and
(75) Inventors/Applicants (for US only): WINTER, Greg
[GB/GB]; MRC Laboratory of Molecular Biology, Hills
Road, Cambridge CB2 2QH (GB). TOMLINSON, Ian
[GB/GB]; Domantis Limited, Granta Park, Abington,
Cambridge CB1 6GS (GB). IGNATOVICH, Olga
[BY/GB]; Domantis Limited, Granta Park, Abington,
Cambridge CB1 6GS (GB). HOLT, Lucy [GB/GB];
Domantis Limited, Granta Park, Abington, Cambridge
CB1 6GS (GB). DE ANGELIS, Elena [IT/GB]; Do-
mantis Limited, Granta Park, Abington, Cambridge CB1
6GS (GB). JONES, Phil [GB/GB]; 15 Impington Lane,
Impington, Cambridge CB4 9LT (GB).
(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
European patent (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
ES, FI, FR, GB, GR, HU, IE, IT, LU, MC, NL, PT, RO,
SE, SI, SK, TR), OAPI patent (BF, BJ, CF, CG, CI, CM,
GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
Declaration under Rule 4.17:
— of inventorship (Rule 4.17 (iv)) for US o nly
Published:
— with international search report
— before the expiration of the time limit for amending the
claims and to be republished in the event of receipt of
amendments
(88) Date of publication of the international search report:
10 September 2004
For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.
<
as
fs| (54) Title: IMMUNOGLOBIN SINGLE VARIANT ANTIGEN-BINDING DOMAINS AND DUAL-SPECIFIC CONSTRUCTS
O
(57) Abstract: The invention provides a dual-specific ligand comprising a first immunoglobulin variable domain having a first bind-
ing specificity and a complementary or non-complementary immunoglobulin variable domain having a second binding specificity.
INTERNATIONAL SEARCH REPORT
lnt< a! Application No
P( 5 03/02804
A. CLASSIFICATION OF SUBJECT MATTER ,
IPC 7 C07K16/24 C07K16/18 C07K16/28 A61K39/395 C07K16/46
C12N15/63 C12N15/62 C12N15/13
According to International Patent Classification (IPC) or to both national classification and IPC
B. FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols)
IPC 7 C07K
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Electronic data base consulted during the international search (name of data base and, where practical, search terms used)
EPO-Internal , BIOSIS, HP I Data, PAJ, MEDLINE, EMBASE
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
A
X
ELS CONRATH K ET AL: "Camel single-domain
antibodies as modular building units in
bi specific and bivalent antibody
construct s "
JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN
SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE,
MD, US,
vol. 276, no. 10,
9 March 2001 (2001-03-09), pages
7346-7350, XP002248402
ISSN: 0021-9258
cited 1n the application
page 7350
page 7349
1-21,
47-56 ,
60,111,
112
113-117
24-26,
38,40,
47-56,
64-67 ,
70-72,
)( Further documents are listed in the continuation of box C.
El
Patent family members are listed in annex.
° Special categories of cited documents :
•A" document defining the general state of the art which is not
considered to be of particular relevance
*E' earlier document but published on or after the international
filing date
*L" document which may throw doubts on priority claim(s) or
which is cited to establish the publication date of another
citation or other special reason (as specified)
■O" document referring to an oral disclosure, use, exhibition or
other means
"P" document published prior to the international filing date but
later than the priority date claimed
"T J iater document published after the international filing date
or priority date and not in conflict with the application but
cited to understand the principle or theory underlying the
invention
'X' document of particular relevance; the claimed invention
cannot be considered novel or cannot be considered to
involve an inventive step when the document is taken alone
"Y 1 document of particular relevance; the claimed invention
cannot be considered to involve an inventive step when the
document is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
in the art.
document member of the same patent family
Date of the actual completion of the international search
8 June 2004
Date of mailing of the international search report j
08. 07. 2004
Name and mailing address of the ISA
European Patent Office, P.B. 5818 Patentlaan 2
NL - 2280 HV Rijswijk
Tel. (+31 -70) 340-2040, Tx, 31 651 epo nJ,
Fax: (+31-70) 340-3016
Authorized officer
Wagner , R
Form PCT/ISA/210 (second shaet) (January 2004)
naoe 1 of 5
INTERNATIONAL SEARCH REPORT
Intc nat Application No
PCl/bB 03/02804
C.(Contlnuation) DOCUMENTS CONSIDERED TO BE RELEVANT
Category °
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
111,112
Y
page 7349
27,28,
36-38,
40,
47-56,
64-67,
70-72,
111,112
page 7349
X
WO 00/29004 A (PEPT0R LTD ;PLAKSIN DANIEL
22,23,
(ID) 25 May 2000 (2000-05-25)
25,
29-31,
38,40,
47-56,
61-63,
70-72,
111,112
example 2
X
REITER Y ET AL: "An antibody
22,23,
single-domain phage display library of a
25,
native heavy chain variable region:
29-31,
isolation of functional single-domain VH
38,40,
molecules with a unique interface"
47-56,
JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB,
61—63 ,
vol. 290, no. 3,
70-72,
l January 1999 (1999-01-01), pages
1*1*1 11 <*s
111 ,112
685-698, XP004461990
ISSN: 0022-2836
page 687
P , A
WO 03/002609 A (MEDICAL RES COUNCIL
41,42,
;T0MLINSON IAN (GB); WINTER GREG (GB);
82 ? 83
IGNATOV) 9 January 2003 (2003-01-09)
page 29, line 8-15
example 6
P,X
WO 03/035694 A (MUYLDERrlANS SERGE ; vLAAMS
22, 113
INTEKUNI V INST BIOTECH (BE))
"1 tn ~. * , onn'i / Anno r*i~ r* -i \
1 May 2003 (2003-05-01)
page 21; example 6
Y
W0 97/30084 A (GENETICS INST)
24-26,
21 August 1997 (1997-08-21)
32-35,
38,40,
47-56,
64-67,
70-72 ,
111,112
example 6
-/—
Form PCT/ISA/210 (continuation of second sheet) (January 2004)
page 2 of 5
INTERNATIONAL SEARCH REPORT
tnt< anal Application No
PL . , iB 03/02804
C.(Continuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT
Category 0
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
v
X
Lr U oOo Oo4 A ^MtUlLAL Kto LUUNL1L;
9Q
oy ,
i/r Mr*\/ i oon Moon nci
io nay iyyu v,iyyu-ub— 10;
A 7 E£
4/— bo ,
111 1 1 9
1 1 1 , 1 1£
Y
example y
O A OC
o^-ob ,
o o /in
oo ,4U ,
A 7 C£
4/-bo ,
04-0/ ,
70-72,
111,112
Y
example b
LI ,Zo\
oo-oo,
4U 3
4/-50,
o4-67,
7r\ 7 0
111 119
111, 1 l£
Y
— — _
WO 98/40469 A (MCFARLAND CLIVE DAVID
27,28,
. 1 IMnTDLIAnn datdtpta ammc /aii>. r&DHT at rpf»
; UNUtKWUUD r A 1 Kit 1 A ANNh t AU J ; UAKU1AL LKL
oO— oo ,
Nj 1/ oeptemoer iyyo \, iyyo-uy-i/ j
/in
4U,
/17 C£
4/— bo ,
04—0/ ,
7fl— 79
/ U"~/ c ,
111 119
111,11c
page o
A
SMITH BRYAN J ET AL: "Prolonged in vivo
1-21,
residence times or antioody fragments
4/— bo ,
associated witn aiDumin
£H 111
OU ,111,
D T Af AM 11 IP A TIT PUrMTCTDV
119
1 1*1
vol. iz, no. o ? September /uui (.zuui-uyj,
»~ ... rt.^. r» 7CA 7C£ VDnn997n7^ 1
pacjes /bU-/bo 5 ArUu&£/u/ol
ISdN: 1U4d-1oUZ
tne wnoie document
ft
A
VAN uhiv DLutKhrJ 1 ti AL: Duiiuing novel
1— cl ,
binding Ugands to B7.1 and B7.2 based on
47-56,
human antibody single variable light chain
60,111,
domains"
112
JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB,
vol. 310, no. 3,
13 July 2001 (2001-07-13), pages 591-601,
XP004464206
ISSN: 0022-2836
abstract
-/—
Form PCT/ISA7210 (coniinuation of second sheet) (January 2004]
oaae 3 of 5
INTERNATIONAL SEARCH REPORT
Inte >na! Application No
PC./JB 03/02804
C.(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT
Category °
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
A
■ a* ■ ■ \ # 1 f"*v ft ■ ft ft 1 B^H B^BV m » P * • B BY T • ~1 ■ *
MUYLDERMANS S ET AL: UNIQUE
1-21,
SINGLE-DOMAIN ANTIGEN BINDING FRAGMENTS
J* l ft ■%■» ftp' ^/ 1 If l •** ■ ^ * l • ft l *4U *— . 1 V 1^ aJU I % law* Jtm 1 ft wl 1 I ft 1 1 ftp! ■ 1 • ■ 1 ftp*
47-56
1 / aV V/ ,
DERIVES FROM NATURALLY OCCURRING CAMEL
60,111,
* ■ tP"* A 1 ||f i|J| VMK » | ft ft ■ w^"» PVP ft** PK VIM |M -a- a a
HEAVY-CHAIN ANTIBODIES"
112
JOURNAL OF MOLECULAR RECOGNITION, HEYDEN &
-h. A. ft | 1 Mi Vft. | _a**a, ft » pK W^ ft * jk PK
SON LTD. , LONDON , GB ,
vol. 12, no. 2, March 1999 (1999-03),
pages 131-140, XP009012180
ISSN: 0952-3499
>!■ S»* 1 » • ftp* -p* W 4— 1 *p» «pr
abstract
A
RIECHMANN L ET AL: "Single domain
1-21,
antibodies: Comparison of camel VH and
47-56,
Pa p. a a * a * ■ ■ a-
camel ised human VH domains"
60,111,
BIOSIS,
112
XP004187632
the whole document
A
HOLLIGER P ET AL: "RETARGETING SERUM
1-21,
■as a ■ a a 1 afth ^Bk — a aa. a la ppa ■> * ■ vaft bbbb a ■ aaw am — aaM ^a. aaa* «■> hi — ass aa aa. ^bk Ml aai aaaa ^Ba. ■ ■
IMMUNOGLOBULIN WITH BISPECIFIC DIABODIES
47-56,
| 1 M ■» ■ | 1 *■* P— b*k Ban _«a_ T* f™ 1 1 ft 1 1 -~s A \ / ft ft At -ft** | a K. paaa T . — , a s— a a -w- ft K A
NATURE BIOTECHNOLOGY, NATURE PUBLISHING,
60,111,
US,
112
vol. 15, July 1997 (1997-07), pages
632-636, XP002921893
*T» i**a a^%> El 1 <M ^a. — T <PV f« p— a
ISSN: 1087-0156
abstract
A
US 5 644 034 A (RATHJEN DEBORAH ANN ET
22 23
AL) 1 July 1997 (1997-07-01)
25,
29-31,
38,40,
47-56,
61-63,
70-72,
111,112
column 2
A
WO 91/02078 A (PEPTIDE TECHNOLOGY LTD)
■ ■ ft^ ah g *■ ~* # ftBP" • 1 \ 1 tpM | 1 Bate lw Lai i J HP* ftp" ■ lift ^af ftaa XaF ^P« I Ea— 1 «— ' #
22 23
21 February 1991 (1991-02-21)
25,
29-31,
38,40,
47-56,
61-63,
70-72,
111,112
page 4, line 3,19
-/--
Form PCT/ISA/210 (continuation of second sheet) (January 2004)
page 4 of 5
INTERNATIONAL SEARCH REPORT
In 'ionat Application No
PCT/GB 03/02804
C.(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT
Category
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
A
P,A
TANHA J ET AL: "Optimal design features
of camel i zed human single-domain antibody
1 i for sir i g s rf
THE JOURNAL OF BIOLOGICAL CHEMISTRY.
UNITED STATES 6 JUL 2001,
vol . 276, no. 27,
6 July 2001 (2001-07-06), pages
24774-24780, XP002283749
ISSN: 0021-9258
the whole document
WO 02/072141 A (HERMAN WILLIAM)
19 September 2002 (2002-09-19)
the whole document
1-117
1-117
Form PCT/tSA/210 (continuation of second sheet) (January 2004-)
page 5 of 5
INTERNATIONAL SEARCH REPORT
srnational application No.
PCT/GB 03/02804
Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet)
This international Search Report has not been established in respect of certain claims under Article 17(2) (a) for the following reasons:
1. Claims Nos.:
because they relate to subject matter not required to be searched by this Authority, namely:
2.
Claims Nos.:
because they relate to parts of the International Application that do not comply with the prescribed requirements to such
an extent that no meaningful International Search can be carried out, specifically:
3. I | Claims Nos.:
because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).
Box (I Observations where unity of invention is Sacking (Continuation of item 2 of first sheet)
This International Searching Authority found multiple inventions in this international application, as follows:
see additional sheet
X
As ali required additional search fees were timely paid by the applicant, this International Search Report covers all
searchable claims.
2. | | As all searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment
of any additional fee.
3. | 1 As only some of the required additional search fees were timely paid by the applicant, this International Search Report
' 1 covers only those claims for which fees were paid, specifically claims Nos.:
4. | | No required additional search fees were timely paid by the applicant. Consequently, this International Search Report is
restricted to the invention first mentioned in the claims; it is covered by claims Nos.:
Remark on Protest | | The additional search fees were accompanied by the applicant's protest.
[ X | No Protest accompanied the payment of additional search fees.
Form PCT/ISA/210 (continuation of first sheet (1)) (July 1998)
Internationa! Application No. PCT7 GB 03/02804
FURTHER INFORMATION CONTINUED FROM PCT/ISA/ 210
This International Searching Authority found multiple (groups of)
inventions in this international application, as follows:
1. Claims: 1-7,8-10,11-21,47-56 (all 10 in part),
60 (in part), 111-112 (both in part)
Ligand comprising a first immunoglobulin variable domain
having a first antigen or epitope specificity and a second
immunoglobulin variable domain having a second antigen or
epitope specificity wherein one or both of the antigens or
epitopes acts to increase the half-life of the ligand in
vivo, and the variable domains are not complementary to one
another.
2. Claims: 22,23,25,29-31, 38 (in part) ,40,
47-56 (all 10 part), 61-63, 70-72 (all 3 in part),
111-112 (both in part)
Single domain antibody monomer ligand specific for TNF- .
3. Claims: 24,26,25,32-35, 38 (in part), 40,
47-56 (all 10 in part), 64-67,
70-72 (all 3 in part), 111-112 (both in part)
Single domain antibody monomer ligand specific for TNF
receptor 1.
4. Claims: 27, 28, 36,37, 38 (in part)40,
47-56 (all 10 in part), 64-67,
70-72 (all 3 in part), 111-112 (both in part)
Single domain antibody monomer ligand specific for serum
albumin.
5. Claims: 39, 47-56 (all 10 in part),
111-112 (both in part).
Single domain antibody monomer ligand comprising a terminal
Cys residue.
6. Claims: 41-46, 47-56 (all 10 in part), 60 (in part).
A dual specific ligand comprising at least a single domain
antibody monomer according to inventions 2-5.
page 1 of 2
International Application No. PC7Y GB 03/02804
FURTHER INFORMATION CONTINUED FROM PCT/ISA/ 210
7. Claims: 73-81, 82-104, 105-110
A closed conformation multi specif ic ligand comprising a
first epitope binding domain having a first epitope binding
specificity and a non-complementary second epitope binding
domain having a second epitope binding specificity, wherein
the first and second binding specificities are capable of
competing for epitope binding such that the closed
conformation multi-specific ligand cannot bind both
epitopes simultaneously. Methods for producing the closed
conformation multi specific ligand. Methods using the closed
conformation multi specif ic ligands.
8. Claims: 113-117
Method for preparing a chelating mul timeric ligand, specific
for adjacent epitopes.
page 2 of 2
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WO 0029004 A
25-05-2000
AU
765201
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AU
6486999
A
05-06-2000
a a
CA
2351669
A 1
Al
P> p ftr p>/"> p> r>
25-05-2000
EP
1131079
Al
12-09-2001
WO
0029004
Al
25-05-2000
KIT
HZ
IT "1 *f A fC /2
511466
A
O P\ P> A o o r\ o
29-04-2003
US
2002012909
Al
31-01-2002
W0 03002609 A
09-01-2003
r\ a
CA
2447851
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09-01-2003
EP
1399484
ft A
A2
24-03-2004
WO
AOAftft/^Aft
03002609
ft a
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09-01-2003
W0 03035694 A
01-05-2003
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1306386
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02-05-2003
WO
ft ft i - p* P"l >|
03035694
A2
ft"i ftr" •ftftrtft
01-05-2003
W0 9730084 A
21-08-1997
us
5843675
A
01-12-1998
1 1 a
US
r ~j h ft ft O 1
5712381
A
27-01-1998
All
AU
O ft ft /"V *T
2268697
A
ft ft Art *1 A ft T
02-09-1997
WO
9730084
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fti p\ p\ -i pv pv —y
21-08-1997
1 IP
us
6322972
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A"7 11 AAAt
27-11-2001
us
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5891675
ii
A
06-04-1999
1 IP
us
2002164716
Al
AT f "1 AAAA
07-11-2002
EP 0368684 A
16-05-1990
ft T*
AT
"1 ft P> P** O "1
102631
T
1 r* AO "f AA A
15-03-1994
a l l
AU
634186
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18-02-1993
nil
AU
4520189
A
AA A r~ 1 AAA
28-05-1990
CA
ps p\ ^ p« p» p* pi
2002868
a -i
Al
"1 "1 A r~ "1 AAA
11-05-1990
DE
>C f> P^i "I o p* r* P>
689 I 3658
Dl
1/1 ft Jt 1 ftft A
14-04-1994
rv r"
DE
pop ^ « p» r" ft
68913658
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08-09-1994
DK
164790
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ft "T ft Pv "J Aft/"»
07-09-1990
EP
ft o /- t~\ f A n
0368684
Al
1 fT A P* "1 AAA
16-05-1990
ES
nnp rtPT7
2052027
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T3
ft -1 ft"T 1Aft^
01-07-1994
wo
9005144
Al
*IT AP* 1 AAA
17-05-1990
JP
2919890
B2
19-07-1999
JP
3502801
T
27-06-1991
KR
184860
D 1
A "1 A /> 1 AAA
01-04-1999
MO
903059
A
07-09-1990
US
2003130496
Al
10-07-2003
US
pi pv r> o *i "i yi /*rA
2003114659
ft -1
Al
"1 A ft /T OAAA
19-06-2003
us
p* pi p> r~ i p*
6248516
Bl
19-06-2001
us
/p r— « t— -i /J pj
6545142
Bl
r> p\ ft ft OAAA
08-04-2003
W0 9840469 A
17-09-1998
All
AU
P P P P "T A A
6602798
A
29-09-1998
1 1 o
WO
9840469
Al
-i — r pi p> i p\ p\ pv
17-09-1998
US 5644034 A
01-07-1997
us
pi pn <*\ w oft.r""n
2003139577
Al
ftft r\'~~l A A A A
24-07-2003
1 1 a
us
ft p» p> ft -1 r\ AT O ft
2003139580
Al
AVI AT AAAO
24-07-2003
us
ftftftA 1
2003162948
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AA AA OAAO
28-08-2003
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2003135029
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17-07-2003
1 J o
us
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2003199678
Al
AA 1 A OAAO
23-10-2003
us
2003232970
Al
18-12-2003
us
2003170204
Al
11-09-2003
us
2003208049
Al
06-11-2003
us
2004002588
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01-01-2004
us
2003171553
Al
11-09-2003
us
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04-09-2003
us
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Al
20-11-2003
us
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Form PCT/ISA/210 (patent family annex) (January 2004)
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US 5644034
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US
2003208047
Al
06-11-2003
US
2003171554
Al
11-09-2003
US
2003171555
Al
11-09-2003
US
2004002590
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01-01-2004
US
2004092721
Al
13-05-2004
US
2003225254
Al
04-12-2003
US
6416757
Bl
09-07-2002
US
2003232971
Al
18-12-2003
US
6593458
Bl
15-07-2003
US
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US
2001018508
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30-08-2001
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5959087
A
28-09-1999
AU
640400
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26-08-1993
AU
6145490
A
11-03-1991
WO
9102078
Al
21-02-1991
CA
2064915
Al
08-02-1991
DE
690Z7121
Dl
ci— uo-iyyo
DE
69027121
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14-11-1996
DK
486526
T3
24-06-1996
EP
0486526
Al
27-05-1992
JP
3443119
B2
02-09-2003
JP
JP
2003096096
2003231699
A
A
03-04-2003
19-08-2003
WO 9102078
A
21-02-1991
AU
640400
B2
26-08-1993
AU
6145490
A
11-03-1991
WO
9102078
Al
21-02-1991
EP
0486526
Al
27-05-1992
CA
2064915
Al
08-02-1991
DE
69027121
Dl
27-06-1996
DE
69027121
T2
14-11-1996
DK
486526
T3
24-06-1996
JP
3443119
B2
02-09-2003
JP
2003096096
A
03-04-2003
JP
2003231699
A
19-08-2003
US
2003162948
Al
28-08-2003
US
2003232970
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18-12-2003
US
2004002588
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01-01-2004
US
2003171553
Al
11-09-2003
US
2003166874
Al
04-09-2003
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2003216552
Al
20-11-2003
US
2004002589
Al
01-01-2004
US
2003208047
Al
06-11-2003
US
5644034
A
01-07-1997
US
2003171554
Al
11-09-2003
US
2003171555
Al
11-09-2003
US
2004002590
Al
01-01-2004
us
2004092721
Al
13-05-2004
U4— id.—cUU o
us
6416757
Bl
09-07-2002
us
2003232971
Al
18-12-2003
us
6593458
Bl
15-07-2003
us
2001018507
Al
30-08-2001
us
2001023287
Al
20-09-2001
us
2001018508
Al
30-08-2001
W0 02072141
A
19-09-2002
wo
02072141
A2
19-09-2002
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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
CORRECTED VERSION
(19) World Intellectual Property Organization
International Bureau
(43) International Publication Date
8 January 2004 (08.01.2004)
PCT
(10) International Publication Number
WO 2004/003019 A3
(51) International Patent Classification : C07K 16/24,
16/18, 16/28, A61K 39/395, C07K 16/46, C12N 15/63,
15/62, 15/13
(21) International Application Number:
PCT/GB2003/002804
(22) International Filing Date: 30 June 2003 (30.06.2003)
(25) Filing Language:
(26) Publication Language:
English
English
(30) Priority Data:
PCT/GB02/03014 28 June 2002 (28.06.2002) GB
0230202.4 27 December 2002 (27.12.2002) GB
(7 1) Applicant (fo r all designated States except US ) : DOMAN-
TIS LIMITED [GB/GB]; 315 Cambridge Science Park,
Cambridge, Cambridgeshire CB4 0WG (GB).
(72) Inventors; and
(75) Inventors/Applicants (for US only): WINTER, Greg
[GB/GB]; MRC Laboratory of Molecular Biology, Hills
Road, Cambridge CB2 2QH (GB). TOMLINS ON, Ian
[GB/GB]; Domantis Limited, Granta Park, Abington,
Cambridge CB1 6GS (GB). IGNATOVICH, Olga
[BY/GB]; Domantis Limited, Granta Park, Abington,
Cambridge CB1 6GS (GB). HOLT, Lucy [GB/GB];
Domantis Limited, Granta Park, Abington, Cambridge
CB1 6GS (GB). DE ANGELIS, Elena [IT/GB]; Domantis
Limited, Granta Park, Abington, Cambridge CB1 6GS
(GB). JONES, Phillip [GB/GB]; 15 Impington Lane,
Impington, Cambridge CB4 9LT (GB).
(74) Agents: MASCHIO, Antonio et al.; D. Young & Co., 120
Holborn, London EC1N 2DY (GB).
(81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
CZ, DE, DK, DM, DZ, EC, EE, ES, FT, GB, GD, GE, GH,
GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
MX, MZ, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RU, SC,
SD, SE, SG, SK, SL, SY, TJ, TM, TN, TR, TT, TZ, UA,
UG, US, UZ, VC, VN, YU, ZA, ZM, ZW.
(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ,
TM), European patent (AT, BE, BG, CH, CY, CZ, DE,
DK, EE, ES, FI, FR, GB, GR, HU, IE, IT, LU, MC, NL,
PT, RO, SE, SI, SK, TR), OAPI patent (BF, BJ, CF, CG,
CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
Declaration under Rule 4.17:
— of invento rship ( Rule 4. 1 7( iv ) )
Published:
— with international search report
(88) Date of publication of the international search report:
10 September 2004
(48) Date of publication of this corrected version:
27 April 2006
(15) Information about Correction:
see PCT Gazette No. 17/2006 of 27 April 2006
For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.
<
OS
(54) Title: IMMUNOGLOBIN SINGLE VARIANT ANTIGEN-BINDING DOMAINS AND DUAL-SPECIFIC CONSTRUCTS
(57) Abstract: The invention provides a dual-specific ligand comprising a first immunoglobulin variable domain having a first
binding specificity and a complementary or non-complementary immunoglobulin variable domain having a second binding speci-
ficity.
o
WO 2004/003019
PCT/GB2003/002804
Ligand
The present invention relates to dual specific ligands. In particular, the invention
provides a method for the preparation of dual-specific ligands comprising a first
5 immunoglobulin single variable domain binding to a first antigen or epitope, and a second
immunoglobulin single variable domain binding to a second antigen or epitope. More
particularly, the invention relates to dual-specific ligands wherein binding to at least one
of the first and second antigens or epitopes acts to increase the half-life of the ligand in
vivo. Open and closed conformation ligands comprising more than one binding specificity
10 are described.
Introduction
The antigen binding domain of an antibody comprises two separate regions: a heavy
15 chain variable domain (Vr) an d a. light chain variable domain (v L : which can be either
V K or Vx,). The antigen binding site itself is formed by six polypeptide loops: three from
V H domain (HI, H2 and H3) and three from v L domain (LI, L2 and L3). A diverse
primary repertoire of V genes that encode the Vh an d Vl domains is produced by the
combinatorial rearrangement of gene segments. The Vh g ei * e is produced by the
20 recombination of three gene segments, Vh> ^ anc * J H- hi humans, there are approximately
51 functional Vh segments (Cook and Tomlinson (1995) Immunol Today, 16: 237), 25
functional D segments (Corbett et ah (1997) J. Moh Bioh, 268: 69) and 6 functional Jh
segments (Ravetch et ah (1981) Cell, 27: 583), depending on the haplotype. The Vh
segment encodes the region of the polypeptide chain which forms the first and second
25 antigen binding loops of the Vh domain (HI and H2), whilst the y H , D an d ^H segments
combine to form the third antigen binding loop of the Vh domain (H3). The Vl g e ^e is
produced by the recombination of only two gene segments, Vl an d JL- hi humans, there
are approximately 40 functional V K segments (Schable and Zachau (1993) Bioh Chem.
Hoppe-Seyler, 374: 1001), 31 functional Y% segments (Williams et ah (1996) J. Moh
30 Bioh, 264: 220; Kawasaki et ah (1997) Genome Res., 7: 250), 5 functional J K segments
(Hieter et ah (1982) J. Bioh Chem., 257: 1516) and 4 functional segments (Vasicek
WO 2004/003019 PCT/GB2003/002804
2
and Leder (1990) J. Exp. Med., 172: 609), depending on the haplotype. The v L segment
encodes the region of the polypeptide chain which forms the first and second antigen
binding loops of the v L domain (LI and L2), whilst the v L and J L segments combine to
form the third antigen binding loop of the v L domain (L3). Antibodies selected from this
5 primary repertoire are believed to be sufficiently diverse to bind almost all antigens with
at least moderate affinity. High affinity antibodies are produced by "affinity maturation"
of the rearranged genes, in which point mutations are generated and selected by the
immune system on the basis of improved binding.
10 Analysis of the structures and sequences of antibodies has shown that five of the six
antigen binding loops (HI, H2, LI, L2, L3) possess a limited number of main-chain
conformations or canonical structures (Chothia and Lesk (1987) J, Mol Biol, 196: 901;
Chothia et al (1989) Nature, 342: 877). The main-chain conformations are determined by
(i) the length of the antigen binding loop, and (ii) particular residues, or types of residue,
15 at certain key position in the antigen binding loop and the antibody framework. Analysis
of the loop lengths and key residues has enabled us to the predict the main-chain
conformations of HI, H2, LI, L2 and L3 encoded by the majority of human antibody
sequences (Chothia et al (1992) J. Mol Biol, 221: 799; Tomlinson et al (1995) EMBO
J., 14: 4628; Williams et al (1996) J. Mol Biol, 264: 220). Although the H3 region is
20 much more diverse in terms of sequence, length and structure (due to the use of D
segments), it also forms a limited number of main-chain conformations for short loop
lengths which depend on the length and the presence of particular residues, or types of
residue, at key positions in the loop and the antibody framework (Martin et al (1996) J.
Mol Biol, 263: 800; Shirai et al (1996) FEBS Letters, 399: 1.
25
Bispecific antibodies comprising complementary pairs of v H and V L regions are known in
the art. These bispecific antibodies must comprise two pairs of v H and V L S > eaclx Vi/Vl
pair binding to a single antigen or epitope. Methods described involve hybrid hybridomas
(Milstein & Cuello AC, Nature 305:537-40), minibodies (Hu et al, (1996) Cancer Res
30 56:3055-3061;), diabodies (Holliger et al, (1993) Proc. Natl. Acad. Sci. USA 90, 6444-
6448; WO 94/13804), chelating recombinant antibodies (CRAbs; (Neri et al, (1995) J.
Mol. Biol. 246, 367-373), biscFv (e.g. Atwell et al, (1996) Mol. Immunol. 33, 1301-
1312), "knobs in holes" stabilised antibodies (Carter et al, (1997) Protein Sci. 6, 781-
WO 2004/003019 PCT/GB2003/002804
3
788). In each case each antibody species comprises two antigen-binding sites, each
fashioned by a complementary pair of v H an d V L domains. Each antibody is thereby able
to bind to two different antigens or epitopes at the same time, with the binding to EACH
antigen or epitope mediated by a Vh an d its complementary Vl domain. Each of these
5 techniques presents its particular disadvantages; for instance in the case of hybrid
hybridomas, inactive V u /y L P airs can greatly reduce the fraction of bispecific IgG.
Furthermore, most bispecific approaches rely on the association of the different wVl
pairs or the association of Vh an d V L chains to recreate the two different Vh^Vl binding
sites. It is therefore impossible to control the ratio of binding sites to each antigen or
10 epitope in the assembled molecule and thus many of the assembled molecules will bind
to one antigen or epitope but not the other. In some cases it has been possible to engineer
the heavy or light chains at the sub-unit interfaces (Carter et al, 1997) in order to improve
the number of molecules which have binding sites to both antigens or epitopes but this
never results in all molecules having binding to both antigens or epitopes.
15
There is some evidence that two different antibody binding specificities might be
incorporated into the same binding site, but these generally represent two or more
specificities that correspond to structurally related antigens or epitopes or to antibodies
that are broadly cross-reactive.. For example, cross-reactive antibodies have been
20 described, usually where the two antigens are related in sequence and structure, such as
hen egg white lysozyme and turkey lysozyme (McCafferty et al., WO 92/01047) or to
free hapten and to hapten conjugated to carrier (Griffiths AD et al. EMBO J 1994 13:14
3245-60). In a further example, WO 02/02773 (Abbott Laboratories) describes antibody
molecules with "dual specificity". The antibody molecules referred to are antibodies
25 raised or selected against multiple antigens, such that their specificity spans more than a
single antigen. Each complementary Vh/V l P air in the antibodies of WO 02/02773
specifies a single binding specificity for two or more structurally related antigens; the v H
and Vl domains in such complementary pairs do not each possess a separate specificity.
The antibodies thus have a broad single specificity which encompasses two antigens,
30 which are structurally related. Furthermore natural autoantibodies have been described
that are polyreactive (Casali & Notkins, Ann. Rev. Immunol. 7, 515-531), reacting with at
least two (usually more) different antigens or epitopes that are not structurally related. It
WO 2004/003019 PCT/GB2003/002804
4
has also been shown that selections of random peptide repertoires using phage display
technology on a monoclonal antibody will identify a range of peptide sequences that fit
the antigen binding site. Some of the sequences are highly related, fitting a consensus
sequence, whereas others are very different and have been termed mimotopes (Lane &
Stephen, Current Opinion in Immunology, 1993, 5, 268-271). It is therefore clear that a
natural four-chain antibody, comprising associated and complementary Vh and y L
domains, has the potential to bind to many different antigens from a large universe of
known antigens. It is less clear how to create a binding site to two given antigens in the
same antibody, particularly those which are not necessarily structurally related.
Protein engineering methods have been suggested that may have a bearing on this. For
example it has also been proposed that a catalytic antibody could be created with a
binding activity to a metal ion through one variable domain, and to a hapten (substrate)
through contacts with the metal ion and a complementary variable domain (Barbas et al.,
1993 Proc. Natl. Acad. Sci USA 90, 6385-6389). However in this case, the binding and
catalysis of the substrate (first antigen) is proposed to require the binding of the metal ion
(second antigen). Thus the binding to the Vf/V L pairing relates to a single but multi-
component antigen.
Methods have been described for the creation of bispecific antibodies from camel
antibody heavy chain single domains in which binding contacts for one antigen are
created in one variable domain, and for a second antigen in a second variable domain.
However the variable domains were not complementary. Thus a first heavy chain variable
domain is selected against a first antigen, and a second heavy chain variable domain
against a second antigen, and then both domains are linked together on the same chain to
give a bispecific antibody fragment (Conrath et al., J. Biol. Chem. 270, 27589-27594).
However the camel heavy chain single domains are unusual in that they are derived from
natural camel antibodies which have no light chains, and indeed the heavy chain single
domains are unable to associate with camel light chains to form complementary y H and
V L pairs.
Single heavy chain variable domains have also been described, derived from natural
antibodies which are normally associated with light chains (from monoclonal antibodies
WO 2004/003019 PCT/GB2003/002804
5
or from repertoires of domains; see EP-A-0368684). These heavy chain variable
domains have been shown to interact specifically with one or more related antigens but
have not been combined with other heavy or light chain variable domains to create a
ligand with a specificity for two or more different antigens . Furthermore, these single
5 domains have been shown to have a very short in vivo half-life. Therefore such domains
are of limited therapeutic value.
It has been suggested to make bispecific antibody fragments by linking heavy chain
variable domains of different specificity together (as described above). The disadvantage
10 with this approach is that isolated antibody variable domains may have a hydrophobic
interface that normally makes interactions with the light chain and is exposed to solvent
and may be "sticky" allowing the single domain to bind to hydrophobic surfaces.
Furthermore, in the absence of a partner light chain the combination of two or more
different heavy chain variable domains and their association, possibly via their
1 5 hydrophobic interfaces, may prevent them from binding to one in not both of the ligands
they are able to bind in isolation. Moreover, in this case the heavy chain variable
domains would not be associated with complementary light chain variable domains and
thus may be less stable and readily unfold (Worn & Pluckthun, 1998 Biochemistry 37,
13120-7).
20
Summary of the invention
The inventors have described, in their copending international patent application WO
03/002609 as well as copending unpublished UK patent application 0230203.2, dual
25 specific immunoglobulin ligands which comprise immunoglobulin single variable
domains which each have different specificities. The domains may act in competition
with each other or independently to bind antigens or epitopes on target molecules.
In a first configuration, the present invention provides a further improvement in dual
30 specific ligands as developed by the present inventors, in which one specificity of the
ligand is directed towards a protein or polypeptide present in vivo in an organism which
can act to increase the half-life of the ligand by binding to it.
WO 2004/003019 PCT/GB2003/002804
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Accordingly, in a first aspect, there is provided a dual-specific ligand comprising a first
immunoglobulin single variable domain having a binding specificity to a first antigen or
epitope and a second complementary immunoglobulin single variable domain having a
binding activity to a second antigen or epitope, wherein one or both of said antigens or
epitopes acts to increase the half-life of the ligand in vivo and wherein said first and
second domains lack mutually complementary domains which share the same specificity,
provided that said dual specific ligand does not consist of an anti-HS A V H domain and an
anti-p galactosidase V K domain. Preferably, that neither of the first or second variable
domains binds to human serum albumin (HSA).
Antigens or epitopes which increase the half-life of a ligand as described herein are
advantageously present on proteins or polypeptides found in an organism in vivo.
Examples include extracellular matrix proteins, blood proteins, and proteins present in
various tissues in the organism. The proteins act to reduce the rate of ligand clearance
from the blood, for example by acting as bulking agents, or by anchoring the ligand to a
desired site of action. Examples of antigens/epitopes which increase half-life in vivo are
given in Annex 1 below.
Increased half-life is useful in in vivo applications of immunoglobulins, especially
antibodies and most especially antibody fragments of small size. Such fragments (Fvs,
disulphide bonded Fvs, Fabs, scFvs, dAbs) suffer from rapid clearance from the body;
thus, whilst they are able to reach most parts of the body rapidly, and are quick to produce
and easier to handle, their in vivo applications have been limited by their only brief
persistence in vivo. The invention solves this problem by providing increased half-life of
the ligands in vivo and consequently longer persistence times in the body of the functional
activity of the ligand.
Methods for pharmacokinetic analysis and determination of ligand half-life will be
familiar to those skilled in the art. Details may be found in Kenneth A et al: Chemical
Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al
Pharmacokinetc analysis: A Practical Approach (1996). Reference is also made to
"Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2 nd Rev. ex
WO 2004/003019 PCT/GB2003/002804
7
edition (1982), which describes pharmacokinetic parameters such as t alpha and t beta
half lives and area under the curve (AUC).
Half lives (tVz alpha and Wi beta) and AUC can be determined from a curve of serum
5 concentration of ligand against time. The WinNonlin analysis package (available from
Pharsight Corp., Mountain View, CA94040, USA) can be used, for example, to model the
curve. In a first phase (the alpha phase) the ligand is undergoing mainly distribution in
the patient, with some elimination. A second phase (beta phase) is the terminal phase
when the ligand has been distributed and the serum concentration is decreasing as the
10 ligand is cleared from the patient. The t alpha half life is the half life of the first phase
and the t beta half life is the half life of the second phase. Thus, advantageously, the
present invention provides a ligand or a composition comprising a ligand according to the
invention having a ta half— life in the range of 15 minutes or more. In one embodiment,
the lower end of the range is 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5
15 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition, or alternatively, a
ligand or composition according to the invention will have a ta half life in the range of up
to and including 12 hours. In one embodiment, the upper end of the range is 11, 10, 9, 8,
7, 6 or 5 hours. An example of a suitable range is 1 to 6 hours, 2 to 5 hours or 3 to 4
hours.
20
Advantageously, the present invention provides a ligand or a composition comprising a
ligand according to the invention having a tp half— life in the range of 2.5 hours or more.
In one embodiment, the lower end of the range is 3 hours, 4 hours, 5 hours, 6 hours, 7
hours, 10 hours , 11 hours, or 12 hours. In addition, or alternatively, a ligand or
25 composition according to the invention has a t(3 half-life in the range of up to and
including 21 days. In one embodiment, the upper end of the range is 12 hours, 24 hours,
2 days, 3 days, 5 days, 10 days, 15 days or 20 days. Advantageously a ligand or
composition according to the invention will have a tp half life in the range 12 to 60 hours.
In a further embodiment, it will be in the range 12 to 48 hours. In a further embodiment
30 still, it will be in the range 12 to 26 hours.
In addition, or alternatively to the above criteria, the present invention provides a ligand
or a composition comprising a ligand according to the invention having an AUC
WO 2004/003019 PCT/GB2003/002804
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value (area under the curve) in the range of 1 mg.min/ml or more. In one embodiment,
the lower end of the range is 5, 10, 15, 20, 30, 100, 200 or 300mg.min/ml. In addition, or
alternatively, a ligand or composition according to the invention has an AUC in the range
of up to 600 mg.min/ml. In one embodiment, the upper end of the range is 500, 400, 300,
5 200, 150, 100, 75 or 50 mg.min/ml. Advantageously a ligand according to the invention
will have a AUC in the range selected from the group consisting of the following: 15 to
150mg.min/ml, 15 to 100 mg.min/ml, 15 to 75 mg.min/ml, and 15 to 50mg.min/ml.
In a first embodiment, the dual specific ligand comprises two complementary variable
10 domains, i.e. two variable domains that, in their natural environment, are capable of
operating together as a cognate pair or group even if in the context of the present
invention they bind separately to their cognate epitopes. For example, the complementary
variable domains may be immunoglobulin heavy chain and light chain variable domains
(V H and V L ). V H and V L domains are advantageously provided by scFv or Fab antibody
15 fragments. Variable domains may be linked together to form multivalent ligands by, for
example: provision of a hinge region at the C-terminus of each V domain and disulphide
bonding between cysteines in the hinge regions; or provision of dAbs each with a cysteine
at the C-terminus of the domain, the cysteines being disulphide bonded together; or
production of V-CH & V-CL to produce a Fab format; or use of peptide linkers (for
20 example Gly 4 Ser linkers discussed hereinbelow) to produce dimers, trimers and further
multimers.
The inventors have found that the use of complementary variable domains allows the two
domain surfaces to pack together and be sequestered from the solvent. Furthermore the
25 complementary domains are able to stabilise each other. In addition, it allows the creation
of dual-specific IgG antibodies without the disadvantages of hybrid hybridomas as used
in the prior art, or the need to engineer heavy or light chains at the sub-unit interfaces.
The dual-specific ligands of the first aspect of the present invention have at least one
Vh/Vl pair. A bispecific IgG according to this invention will therefore comprise two
30 such pairs, one pair on each arm of the Y-shaped molecule. Unlike conventional
bispecific antibodies or diabodies, therefore, where the ratio of chains used is
determinative in the success of the preparation thereof and leads to practical difficulties,
the dual specific ligands of the invention are free from issues of chain balance. Chain
WO 2004/003019 PCT/GB2003/002804
9
imbalance in conventional bi-specific antibodies results from the association of two
different V L chains with two different V H chains, where V L chain 1 together with V H
chain 1 is able to bind to antigen or epitope 1 and V L chain 2 together with V H chain 2 is
able to bind to antigen or epitope 2 and the two correct pairings are in some way linked to
5 one another. Thus, only when V L chain 1 is paired with V H chain 1 and V L chain 2 is
paired with V H chain 2 in a single molecule is bi-specificity created. Such bi-specific
molecules can be created in two different ways. Firstly, they can be created by association
of two existing V H /V L pairings that each bind to a different antigen or epitope (for
example, in a bi-specific IgG). In this case the V H /V L pairings must come all together in a
10 1:1 ratio in order to create a population of molecules all of which are bi-specific. This
never occurs (even when complementary CH domain is enhanced by "knobs into holes"
engineering) leading to a mixture of bi-specific molecules and molecules that are only
able to bind to one antigen or epitope but not the other. The second way of creating a bi-
specific antibody is by the simultaneous association of two different Vh chain with two
15 different V L chains (for example in a bi-specific diabody). In this case, although there
tends to be a preference for V L chain 1 to pair with V H chain 1 and Vl chain 2 to pair with
Vh chain 2 (which can be enhanced by "knobs into holes" engineering of the V L and V H
domains), this paring is never achieved in all molecules, leading to a mixed formulation
whereby incorrect pairings occur that are unable to bind to either antigen or epitope.
20
Bi-specific antibodies constructed according to the dual-specific ligand approach
according to the first aspect of the present invention overcome all of these problems
because the binding to antigen or epitope 1 resides within the Vh or Vl domain and the
binding to antigen or epitope 2 resides with the complementary Vl or Vh domain,
25 respectively. Since V H and V L domains pair on a 1:1 basis all V H /V L pairings will be bi-
specific and thus all formats constructed using these V H /V L pairings (Fv, scFvs, Fabs,
minibodies, IgGs etc) will have 100% bi-specific activity.
In the context of the present invention, first and second "epitopes" are understood to be
30 epitopes which are not the same and are not bound by a single monospecific ligand. In
the first configuration of the invention, they are advantageously on different antigens, one
of which acts to increase the half-life of the ligand in vivo. Likewise, the first and second
antigens are advantageously not the same.
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The dual specific ligands of the invention do not include ligands as described in WO
02/02773. Thus, the ligands of the present invention do not comprise complementary
V H /V L pairs which bind any one or more antigens or epitopes co-operatively. Instead, the
ligands according to the first aspect of the invention comprise a V h /Vl complementary
pair, wherein the V domains have different specificities.
Moreover, the ligands according to the first aspect of the invention comprise Vh/Vl
complementary pairs having different specificities for non-structurally related epitopes or
antigens. Structurally related epitopes or antigens are epitopes or antigens which possess
sufficient structural similarity to be bound by a conventional V h /Vl complementary pair
which acts in a co-operative manner to bind an antigen or epitope; in the case of
structurally related epitopes, the epitopes are sufficiently similar in structure that they
"fit" into the same binding pocket formed at the antigen binding site of the V H /V L dimer.
In a second aspect, the present invention provides a ligand comprising a first
immunoglobulin variable domain having a first antigen or epitope binding specificity and
a second immunoglobulin variable domain having a second antigen or epitope binding
specificity wherein one or both of said first and second variable domains bind to an
antigen which increases the half-life of the ligand in vivo, and the variable domains are
not complementary to one another.
In one embodiment, binding to one variable domain modulates the binding of the ligand
to the second variable domain.
In this embodiment, the variable domains may be, for example, pairs of V H domains or
pairs of Vl domains. Binding of antigen at the first site may modulate, such as enhance or
inhibit, binding of an antigen at the second site. For example, binding at the first site at
least partially inhibits binding of an antigen at a second site. In such an embodiment, the
ligand may for example be maintained in the body of a subject organism in vivo through
binding to a protein which increases the half-life of the ligand until such a time as it
becomes bound to the second target antigen and dissociates from the half-life increasing
protein.
WO 2004/003019
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Modulation of binding in the above context is achieved as a consequence of the structural
proximity of the antigen binding sites relative to one another. Such structural proximity
can be achieved by the nature of the structural components linking the two or more
5 antigen binding sites, eg by the provision of a ligand with a relatively rigid structure that
holds the antigen binding sites in close proximity. Advantageously, the two or more
antigen binding sites are in physically close proximity to one another such that one site
modulates the binding of antigen at another site by a process which involves steric
hindrance and/or conformational changes within the immunoglobulin molecule.
10
The first and the second antigen binding domains may be associated either covalently or
non-covalently. In the case that the domains are covalently associated, then the
association may be mediated for example by disulphide bonds or by a polypeptide linker
such as (Gly 4 Ser) n , where n = from 1 to 8, eg, 2, 3, 4, 5 or 7.
15
Ligands according to the invention may be combined into non-immunoglobulin multi-
ligand structures to form multivalent complexes, which bind target molecules with the
same antigen, thereby providing superior avidity, while at least one variable domain binds
an antigen to increase the half life of the multimer. For example natural bacterial
20 receptors such as SpA have been used as scaffolds for the grafting of CDRs to generate
ligands which bind specifically to one or more epitopes. Details of this procedure are
described in US 5,831,012. Other suitable scaffolds include those based on fibronectin
and affibodies. Details of suitable procedures are described in WO 98/58965. Other
suitable scaffolds include lipocallin and CTLA4, as described in van den Beuken et aL, J.
25 Mol. Biol. (2001) 310, 591-601, and scaffolds such as those described in WO0069907
(Medical Research Council), which are based for example on the ring structure of
bacterial GroEL or other chaperone polypeptides.
Protein scaffolds may be combined; for example, CDRs may be grafted on to a CTLA4
30 scaffold and used together with immunoglobulin V H or V L domains to form a ligand.
Likewise, fibronectin, lipocallin and other scaffolds may be combined.
WO 2004/003019 PCT/GB2003/002804
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In the case that the variable domains are selected from V~gene repertoires selected for
instance using phage display technology as herein described, then these variable domains
can comprise a universal framework region, such that is they may be recognised by a
specific generic ligand as herein defined. The use of universal frameworks, generic
5 ligands and the like is described in WO99/20749. In the present invention, reference to
phage display includes the use of both phage and/or phagemids.
Where V-gene repertoires are used variation in polypeptide sequence is preferably located
within the structural loops of the variable domains. The polypeptide sequences of either
10 variable domain may be altered by DNA shuffling or by mutation in order to enhance the
interaction of each variable domain with its complementary pair.
In a preferred embodiment of the invention the 'dual-specific ligand' is a single chain Fv
fragment. In an alternative embodiment of the invention, the 'dual-specific ligand 5
15 consists of a Fab region of an antibody. The term "Fab region" includes a Fab-like
region where two VH or two VL domains are used.
The variable regions may be derived from antibodies directed against target antigens or
epitopes. Alternatively they may be derived from a repertoire of single antibody domains
20 such as those expressed on the surface of filamentous bacteriophage. Selection may be
performed as described below.
In a third aspect, the invention provides a method for producing a ligand comprising a
first immunoglobulin single variable domain having a first binding specificity and a
25 second single immunoglobulin single variable domain having a second (different) binding
specificity, one or both of the binding specificities being specific for an antigen which
increases the half-life of the ligand in vivo, the method comprising the steps of:
(a) selecting a first variable domain by its ability to bind to a first epitope,
(b) selecting a second variable region by its ability to bind to a second epitope,
30 (c) combining the variable domains; and
(d) selecting the ligand by its ability to bind to said first epitope and to said second
epitope.
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The ligand can bind to the first and second epitopes either simultaneously or, where there
is competition between the binding domains for epitope binding, the binding of one
domain may preclude the binding of another domain to its cognate epitope. In one
embodiment, therefore, step (d) above requires simultaneous binding to both first and
5 second (and possibly further) epitopes; in another embodiment, the binding to the first
and second epitoes is not simultaneous.
The epitopes are preferably on separate antigens.
10 Ligands advantageously comprise V H /V L combinations, or V H /V H or Vl/V l combinations
of immunoglobulin variable domains, as described above. The ligands may moreover
comprise camelid V H h domains, provided that the V H h domain which is specific for an
antigen which increases the half-life of the ligand in vivo does not bind Hen egg white
lysozyme (HEL), porcine pancreatic alpha-amylase or NmC-A; hcg, BSA-linked RR6 azo
15 dye or S. mutans HG982 cells, as described in Conrath et al, (2001) JBC 276:7346-7350
and W099/23221, neither of which describe the use of a specificity for an antigen which
increases half-life to increase the half life of the ligand in vivo.
In one embodiment, said first variable domain is selected for binding to said first epitope
20 in absence of a complementary variable domain. In a further embodiment, said first
variable domain is selected for binding to said first epitope/antigen in the presence of a
third variable domain in which said third variable domain is different from said second
variable domain and is complementary to the first domain. Similarly, the second domain
may be selected in the absence or presence of a complementary variable domain.
25
The antigens or epitopes targeted by the ligands of the invention, in addition to the half-
life enhancing protein, may be any antigen or epitope but advantageously is an antigen or
epitope that is targeted with therapeutic benefit. The invention provides ligands,
including open conformation, closed conformation and isolated dAb monomer ligands,
30 specific for any such target, particularly those targets further identified herein. Such
targets may be, or be part of, polypeptides, proteins or nucleic acids, which may be
naturally occurring or synthetic. In this respect, the ligand of the invention may bind the
epiotpe or antigen and act as an antagonist or agonist (eg, EPO receptor agonist). One
WO 2004/003019 PCT/GB2003/002804
14
skilled in the art will appreciate that the choice is large and varied. They may be for
instance human or animal proteins, cytokines, cytokine receptors, enzymes co-factors for
enzymes or DNA binding proteins. Suitable cytokines and growth factors include but are
not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor, ENA-78,
Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic, fibroblast growth factor- 10,
FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-pl, insulin, IFN-y,
IGF-I, IGF-II, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77
a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin a,
Inhibin p, IP- 10, keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin,
Mullerian inhibitory substance, monocyte colony inhibitory factor, monocyte attractant
protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-
4, MDC (67 a.a.), MDC (69 a.a.), MIG, MlP-la, MIP-lp, MIP-3a, MIP-3P, MIP-4,
myeloid progenitor inhibitor factor- 1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor,
P-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES,
SDFla, SDFlp, SCF, SCGF, stem cell factor (SCF), TARC, TGF-a, TGF-P, TGF-p2,
TGF-P3, tumour necrosis factor (TNF), TNF-a, TNF-P, TNF receptor I, TNF receptor II,
TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2,
GRO/MGSA, GRO-p, GRO-y, HCC1, 1-309, HER 1, HER 2, HER 3 and HER 4.
Cytokine receptors include receptors for the foregoing cytokines. It will be appreciated
that this list is by no means exhaustive.
In one embodiment of the invention, the variable domains are derived from a respective
antibody directed against the antigen or epitope. In a preferred embodiment the variable
domains are derived from a repertoire of single variable antibody domains.
Ih a further aspect, the present invention provides one or more nucleic acid molecules
encoding at least a dual-specific ligand as herein defined. The dual specific ligand may
be encoded on a single nucleic acid molecule; alternatively, each domain may be encoded
by a separate nucleic acid molecule. Where the ligand is encoded by a single nucleic acid
molecule, the domains may be expressed as a fusion polypeptide, in the manner of a scFv
molecule, or may be separately expressed and subsequently linked together, for example
using chemical linking agents. Ligands expressed from separate nucleic acids will be
linked together by appropriate means.
WO 2004/003019 PCT/GB2003/002804
15
The nucleic acid may further encode a signal sequence for export of the polypeptides
from a host cell upon expression and may be fused with a surface component of a
filamentous bacteriophage particle (or other component of a selection display system)
5 upon expression.
In a further aspect the present invention provides a vector comprising nucleic acid
encoding a dual specific ligand according to the present invention.
10 In a yet further aspect, the present invention provides a host cell transfected with a vector
encoding a dual specific ligand according to the present invention.
Expression from such a vector may be configured to produce, for example on the surface
of a bacteriophage particle, variable domains for selection. This allows selection of
15 displayed variable regions and thus selection of 'dual- specific ligands' using the method
of the present invention.
The present invention further provides a kit comprising at least a dual-specific ligand
according to the present invention.
20
Dual-Specific ligands according to the present invention preferably comprise
combinations of heavy and light chain domains. For example, the dual specific ligand
may comprise a Vh domain and a V L domain, which may be linked together in the form
of an scFv. In addition, the ligands may comprise one or more Ch or Cl domains. For
25 example, the ligands may comprise a ChI domain, C H 2 or C H 3 domain, and/or a C L
domain, Cpl, C|u2, C\i3 or Cjj4 domains, or any combination thereof. A hinge region
domain may also be included. Such combinations of domains may, for example, mimic
natural antibodies, such as IgG or IgM, or fragments thereof, such as Fv, scFv, Fab or
F(ab') 2 molecules. Other structures, such as a single arm of an IgG molecule comprising
30 Vh, Vl, CrI and Cl domains, are envisaged.
In a preferred embodiment of the invention, the variable regions are selected from single
domain V gene repertoires. Generally the repertoire of single antibody domains is
WO 2004/003019 PCT/GB2003/002804
16
displayed on the surface of filamentous bacteriophage. In a preferred embodiment each
single antibody domain is selected by binding of a phage repertoire to antigen.
In a preferred embodiment of the invention each single variable domain may be selected
for binding to its target antigen or epitope in the absence of a complementary variable
region. In an alternative embodiment, the single variable domains may be selected for
binding to its target antigen or epitope in the presence of a complementary variable
region. Thus the first single variable domain may be selected in the presence of a third
complementary variable domain, and the second variable domain may be selected in the
presence of a fourth complementary variable domain. The complementary third or fourth
variable domain may be the natural cognate variable domain having the same specificity
as the single domain being tested, or a non-cognate complementary domain — such as a
"dummy" variable domain.
Preferably, the dual specific ligand of the invention comprises only two variable domains
although several such ligands may be incorporated together into the same protein, for
example two such ligands can be incorporated into an IgG or a multimeric
immunoglobulin, such as IgM. Alternatively, in another embodiment a plurality of dual
specific ligands are combined to form a multimer. For example, two different dual
specific ligands are combined to create a tetra-specific molecule.
It will be appreciated by one skilled in the art that the light and heavy variable regions of
a dual- specific ligand produced according to the method of the present invention may be
on the same polypeptide chain, or alternatively, on different polypeptide chains. In the
case that the variable regions are on different polypeptide chains, then they may be linked
via a linker, generally a flexible linker (such as a polypeptide chain), a chemical linking
group, or any other method known in the art.
In a further aspect, the present invention provides a composition comprising a dual-
specific ligand, obtainable by a method of the present invention, and a pharmaceutically
acceptable carrier, diluent or excipient.
WO 2004/003019 PCT/GB2003/002804
17
Moreover, the present invention provides a method for the treatment and/or prevention
of disease using a 'dual- specific ligand' or a composition according to the present
invention.
5 In a second configuration, the present invention provides multispecific ligands which
comprise at least two non-complementary variable domains. For example, the ligands
may comprise a pair of V H domains or a pair of Vl domains. Advantageously, the
domains are of non-camelid origin; preferably they are human domains or comprise
human framework regions (FWs) and one or more heterologous CDRs. CDRs and
10 framework regions are those regions of an immunoglobulin variable domain as defined in
the Kabat database of Sequences of Proteins of Immunological Interest.
Preferred human framework regions are those encoded by germline gene segments DP47
and DPK9. Advantageously, FW1, FW2 and FW3 of a Vh or Vl domain have the
15 sequence of FW1, FW2 or FWS from DP47 or DPK9. The human frameworks may
optionally contain mutations, for example up to about 5 amino acid changes or up to
about 10 amino acid changes collectively in the human frameworks used in the ligands of
the invention.
20 The variable domains in the multispecific ligands according to the second configuration
of the invention may be arranged in an open or a closed conformation; that is, they may
be arranged such that the variable domains can bind their cognate ligands independently
and simultaneously, or such that only one of the variable domains may bind its cognate
ligand at any one time.
25
The inventors have realised that under certain structural conditions, non-complementary
variable domains (for example two light chain variable domains or two heavy chain
variable domains) may be present in a ligand such that binding of a first epitope to a first
variable domain inhibits the binding of a second epitope to a second variable domain,
30 even though such non-complementary domains do not operate together as a cognate pair.
WO 2004/003019 PCT/GB2003/002804
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Advantageously, the ligand comprises two or more pairs of variable domains; that is, it
comprises at least four variable domains. Advantageously, the four variable domains
comprise frameworks of human origin.
In a preferred embodiment, the human frameworks are identical to those of human
germline sequences.
The present inventors consider that such antibodies will be of particular use in ligand
binding assays for therapeutic and other uses.
Thus, in a first aspect of the second configuration, the present invention provides a
method for producing a multispecific ligand comprising the steps of:
a) selecting a first epitope binding domain by its ability to bind to a first epitope,
b) selecting a second epitope binding domain by its ability to bind to a second
epitope,
c) combining the epitope binding domains; and
d) selecting the closed conformation multispecific ligand by its ability to bind to said
first second epitope and said second epitope.
In a further aspect of the second configuration, the invention provides method for
preparing a closed conformation multi-specific ligand comprising a first epitope binding
domain having a first epitope binding specificity and a non-complementary second
epitope binding domain having a second epitope binding specificity, wherein the first and
second binding specificities compete for epitope binding such that the closed
conformation multi-specific ligand may not bind both epitopes simultaneously, said
method comprising the steps of:
a) selecting a first epitope binding domain by its ability to bind to a first epitope,
b) selecting a second epitope binding domain by its ability to bind to a second
epitope,
c) combining the epitope binding domains such that the domains are in a closed
conformation; and
WO 2004/003019 PCT/GB2003/002804
19
d) selecting the closed conformation multispecific ligand by its ability to bind to
said first second epitope and said second epitope, but not to both said first and
second epitopes simultaneously.
Moreover, the invention provides a closed conformation multi-specific ligand comprising
a first epitope binding domain having a first epitope binding specificity and a non-
complementary second epitope binding domain having a second epitope binding
specificity, wherein the first and second binding specificities compete for epitope binding
such that the closed conformation multi-specific ligand may not bind both epitopes
simultaneously.
An alternative embodiment of the above aspect of the of the second configuration of the
invention optionally comprises a further step (bl) comprising selecting a third or further
epitope binding domain. In this way the multi-specific ligand produced, whether of open
or closed conformation, comprises more than two epitope binding specificities. In a
preferred aspect of the second configuration of the invention, where the multi-specific
ligand comprises more than two epitope binding domains, at least two of said domains are
in a closed conformation and compete for binding; other domains may compete for
binding or may be free to associate independently with their cognate epitope(s).
According to the present invention the term 'multi-specific ligand 5 refers to a ligand
which possesses more than one epitope binding specificity as herein defined.
As herein defined the term 'closed conformation' (multi-specific ligand) means that the
epitope binding domains of the ligand are attached to or associated with each other,
optionally by means of a protein skeleton, such that epitope binding by one epitope
binding domain competes with epitope binding by another epitope binding domain. That
is, cognate epitopes may be bound by each epitope binding domain individually but not
simultaneosuly. The closed conformation of the ligand can be achieved using methods
herein described.
"Open conformation" means that the epitope binding domains of the ligand are attached
to or associated with each other, optionally by means of a protein skeleton, such that
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epitope binding by one epitope binding domain does not compete with epitope binding
by another epitope binding domain.
As referred to herein, the term 'competes' means that the binding of a first epitope to its
5 cognate epitope binding domain is inhibited when a second epitope is bound to its
cognate epitope binding domain. For example, binding may be inhibited sterically, for
example by physical blocking of a binding domain or by alteration of the structure or
environment of a binding domain such that its affinity or avidity for an epitope is reduced.
10 In a further embodiment of the second configuration of the invention, the epitopes may
displace each other on binding. For example, a first epitope may be present on an antigen
which, on binding to its cognate first binding domain, causes steric hindrance of a second
binding domain, or a coformational change therein, which displaces the epitope bound to
the second binding domain.
15
Advantageously, binding is reduced by 25% or more, advantageously 40%, 50%, 60%,
70%o, 80%>, 90% or more, and preferably up to 100% or nearly so, such that binding is
completely inhibited. Binding of epitopes can be measured by conventional antigen
binding assays, such as ELIS A, by fluorescence based techniques, including FRET, or by
20 techniques such as suface plasmon resonance which measure the mass of molecules.
According to the method of the present invention, advantageously, each epitope binding
domain is of a different epitope binding specificity.
25 In the context of the present invention, first and second "epitopes" are understood to be
epitopes which are not the same and are not bound by a single monospecific ligand. They
may be on different antigens or on the same antigen, but separated by a sufficient distance
that they do not form a single entity that could be bound by a single mono-specific V H /V L
binding pair of a conventional antibody. Experimentally, if both of the individual
30 variable domains in single chain antibody form (domain antibodies or dAbs) are
separately competed by a monospecific V H /V L ligand against two epitopes then those two
epitopes are not sufficiently far apart to be considered separate epitopes according to the
present invention.
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The closed conformation multispecific ligands of the invention do not include ligands as
described in WO 02/02773. Thus, the ligands of the present invention do not comprise
complementary Vj/Vl pairs which bind any one or more antigens or epitopes co-
5 operatively. Instead, the ligands according to the invention preferably comprise non-
complementary V H -V H or V L -V L P airs - Advantageously, each v H or V L domain in each
Vh~Vh or Vl~Vl P a ^ r has a different epitope binding specificity, and the epitope binding
sites are so arranged that the binding of an epitope at one site competes with the binding
of an epitope at another site.
10
According to the present invention, advantageously, each epitope binding domain
comprises an immunoglobulin variable domain. More advantageously, each
immunoglobulin variable domain will be either a variable light chain domain (Vl) or a
variable heavy chain domain Vh- I* 1 the second configuration of the present invention,
15 the immunoglobulin domains when present on a ligand according to the present
invention are non-complementary, that is they do not associate to form a Vp/Vl antigen
binding site. Thus, multi-specific ligands as defined in the second configuration of the
invention comprise immunoglobulin domains of the same sub-type, that is either variable
light chain domains (Vl) or variable heavy chain domains (Vh)- Moreover, where the
ligand according to the invention is in the closed conformation, the immunoglobulin
domains may be of the camelid Vhh type.
In an alternative embodiment, the ligand(s) according to the invention do not comprise a
camelid Vhh domain. More particularly, the ligand(s) of the invention do not comprise
one or more amino acid residues that are specific to camelid Vhh domains as compared to
human Vh domains.
Advantageously, the single variable domains are derived from antibodies selected for
binding activity against different antigens or epitopes. For example, the variable domains
may be isolated at least in part by human immunisation. Alternative methods are known
in the art, including isolation from human antibody libraries and synthesis of artificial
antibody genes.
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The variable domains advantageously bind superantigens, such as protein A or protein L.
Binding to superantigens is a property of correctly folded antibody variable domains, and
allows such domains to be isolated from, for example, libraries of recombinant or mutant
domains.
Epitope binding domains according to the present invention comprise a protein scaffold
and epitope interaction sites (which are advantageously on the surface of the protein
scaffold).
Epitope binding domains may also be based on protein scaffolds or skeletons other than
immunoglobulin domains. For example natural bacterial receptors such as SpA have been
used as scaffolds for the grafting of CDRs to generate ligands which bind specifically to
one or more epitopes. Details of this procedure are described in US 5,831,012. Other
suitable scaffolds include those based on fibronectin and affibodies. Details of suitable
procedures are described in WO 98/58965. Other suitable scaffolds include lipocallin and
CTLA4, as described in van den Beuken et al 9 J. Mol. Biol. (2001) 310, 591-601, and
scaffolds such as those described in WO0069907 (Medical Research Council), which are
based for example on the ring structure of bacterial GroEL or other chaperone
polypeptides.
Protein scaffolds may be combined; for example, CDRs may be grafted on to a CTLA4
scaffold and used together with immuno globulin V H or V L domains to form a multivalent
ligand. Likewise, fibronectin, lipocallin and other scaffolds may be combined.
25 It will be appreciated by one skilled in the art that the epitope binding domains of a closed
conformation multispecific ligand produced according to the method of the present
invention may be on the same polypeptide chain, or alternatively, on different polypeptide
chains. In the case that the variable regions are on different polypeptide chains, then they
may be linked via a linker, advantageously a flexible linker (such as a polypeptide chain),
30 a chemical linking group, or any other method known in the art.
10
15
20
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The first and the second epitope binding domains may be associated either covalently or
non-covalently. In the case that the domains are covalently associated, then the
association may be mediated for example by disulphide bonds.
5 In the second configuation of the invention, the first and the second epitopes are
preferably different. They may be, or be part of, polypeptides, proteins or nucleic acids,
which may be naturally occurring or synthetic. In this respect, the ligand of the invention
may bind an epiotpe or antigen and act as an antagonist or agonist (eg, EPO receptor
agonist). The epitope binding domains of the ligand in one embodiment have the same
10 epitope specificity, and may for example simultaneously bind their epitope when multiple
copies of the epitope are present on the same antigen. In another embodiment, these
epitopes are provided on different antigens such that the ligand can bind the epitopes and
bridge the antigens. One skilled in the art will appreciate that the choice of epitopes and
antigens is large and varied. They may be for instance human or animal proteins,
15 cytokines, cytokine receptors, enzymes co-factors for enzymes or DNA binding proteins.
Suitable cytokines and growth factors include but are not limited to: ApoE, Apo-SAA,
BDNF, Cardiotrophin-1, EGF, EGF receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2,
EpoR, FGF-acidic, FGF-basic, fibroblast growth factor- 10, FLT3 ligand, Fractalkine
(CX3C), GDNF, G-CSF, GM-CSF, GF-(31, insulin, IFN-y, IGF-I, IGF-II, IL-la, IL-ip,
20 IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12,
IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Miibin a, Inhibin p, IP-10, keratinocyte growth
factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian inhibitory substance,
monocyte colony inhibitory factor, monocyte attractant protein, M-CSF, MDC (67 a.a.),
MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69
25 a.a.), MIG, MlP-la, MIP-ip, MIP-3a, MIP-3P, MIP-4, myeloid progenitor inhibitor
factor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor, P-NGF, NT-3, NT-4,
Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDFloc, SDFip, SCF,
SCGF, stem cell factor (SCF), TARC, TGF-a, TGF-p, TGF-P2, TGF-p3, tumour necrosis
factor (TNF), TNF-a, TNF-p, TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF,
30 VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-p,
GRO-y, HCC1, 1-309, HER 1, HER 2, HER 3, HER 4, TACE recognition site, TNF BP-I
and TNF BP-II, as well as any target disclosed in Annex 2 or Annex 3 hereto, whether in
combination as set forth in the Annexes, in a different combination or individually.
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Cytokine receptors include receptors for the foregoing cytokines, e.g. IL-1 Rl; IL-6R;
IL-10R; IL-18R, as well as receptors for cytokines set forth in Annex 2 or Annex 3 and
also receptors disclosed in Annex 2 and 3. It will be appreciated that this list is by no
means exhaustive. Where the multispecific ligand binds to two epitopes (on the same or
5 different antigens), the antigen(s) may be selected from this list.
Advantageously, dual specific ligands may be used to target cytokines and other
molecules which cooperate synergistically in therapeutic situations in the body of an
organism. The invention therefore provides a method for synergising the activity of two
10 or more cytokines, comprising administering a dual specific ligand capable of binding to
said two or more cytokines. In this aspect of the invention, the dual specific ligand may
be any dual specific ligand, including a ligand composed of complementary and/or non- •
complementary domains, a ligand in an open conformation, and a ligand in a closed
conformation. For example, this aspect of the invention relates to combinations of V H
15 domains and V L domains, V H domains only and V L domains only.
Synergy in a therapeutic context may be achieved in a number of ways. For example,
target combinations may be therapeutically active only if both targets are targeted by the
ligand, whereas targeting one target alone is not therapeutically effective. In another
20 embodiment, one target alone may provide some low or minimal therapeutic effect, but
together with a second target the combination provides a synergistic increase in
therapeutic effect.
Preferably, the cytokines bound by the dual specific ligands of this aspect of the invention
25 are sleeted from the list shown in Annex 2.
Moreover, dual specific ligands may be used in oncology applications, where one
specificity targets CD89, which is expressed by cytotoxic cells, and the other is tumour
specific. Examples of tumour antigens which may be targetted are given in Annex 3.
30
In one embodiment of the second configuration of the invention, the variable domains are
derived from an antibody directed against the first and/or second antigen or epitope. In a
preferred embodiment the variable domains are derived from a repertoire of single
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variable antibody domains. In one example, the repertoire is a repertoire that is not
created in an animal or a synthetic repertoire. In another example, the single variable
domains are not isolated (at least in part) by animal immunisation. Thus, the single
domains can be isolated from a naive library.
The second configuration of the invention, in another aspect, provides a multi-specific
ligand comprising a first epitope binding domain having a first epitope binding specificity
and a non-complementary second epitope binding domain having a second epitope
binding specificity. The first and second binding specificities may be the same or
different.
In a further aspect, the present invention provides a closed conformation multi-specific
ligand comprising a first epitope binding domain having a first epitope binding specificity
and a non-complementary second epitope binding domain having a second epitope
binding specificity wherein the first and second binding specificities are capable of
competing for epitope binding such that the closed conformation multi-specific ligand
cannot bind both epitopes simultaneously.
In a still further aspect, the invention provides open conformation ligands comprising
non-complementary binding domains, wherein the deomains are specific for a different
epitope on the same target. Such ligands bind to targets with increased avidity.
Similarly, the invention provides multivalent ligands comprising non-complementary
binding domains specific for the same epitope and directed to targets which comprise
multiple copies of said epitope, such as IL-5, PDGF-AA, PDGF-BB, TGF beta, TGF
beta2, TGF beta3 and TNFa, for eample human TNF Receptor 1 and human TNFoe.
In a similar aspect, ligands according to the invention can be configured to bind
individual epitopes with low affinity, such that binding to individual epitopes is not
therapeutically significant; but the increased avidity resulting from binding to two
epitopes provides a theapeutic benefit. In a perticular example, epitopes may be targetted
which are present individually on normal cell types, but present together only on
abnormal or diseased cells, such as tumour cells. In such a situaton, only the abnormal or
WO 2004/003019 PCT/GB2003/002804
26
diseased cells are effectively targetted by the bispecific ligands according to the
invention.
Ligand specific for multiple copies of the same epitope, or adjacent epitopes, on the same
5 target (known as chelating dAbs) may also be trimeric or polymeric (tertrameric or more)
ligands comprising three, four or more non-complementary binding domains. For
example, ligands may be constructed comprising three or four V H domains or V L
domains.
10 Moreover, ligands are provided which bind to multisubunit targets, wherein each binding
domain is specific for a subunit of said target. The ligand may be dimeric, trimeric or
polymeric.
Preferably, the multi-specific ligands according to the above aspects of the invention are
15 obtainable by the method of the first aspect of the invention.
According to the above aspect of the second configuration of the invention,
advantageously the first epitope binding domain and the second epitope binding domains
are non- complementary immunoglobulin variable domains, as herein defined. That is
20 either Vh"V h or V L ~V L variable domains.
Chelating dAbs in particular may be prepared according to a preferred aspect of the
invention, namely the use of anchor dAbs, in which a library of dimeric, trimeric or
multimeric dAbs is constructed using a vector which comprises a constant dAb upstream
25 or downstream of a linker sequence, with a repertoire of second, third and further dAbs
being inserted on the other side of the linker. For example, the anchor or guiding dAb
may be TAR1-5 (Vk), TAR1-27(Vk), TAR2h-5(VH) or TAR2h-6(Vic).
In alternative methodologies, the use of linkers may be avoided, for example by the use of
30 non-covalent bonding or naturall affinity between binding domains such as V H and V K .
The invention accordingly provides a method for preparing a chelating multimeric ligand
comprising the steps of:
WO 2004/003019 PCT/GB2003/002804
27
(a) providing a vector comprising a nucleic acid sequence encoding a single
binding domain specific for a first epitope on a target;
(b) providing a vector encoding a repertoire comprising second binding domains
specific for a second epitope on said target, which epitope can be the same or different to
5 the first epitope, said second epitope being adjacent to said first epitope; and
(c) expressing said first and second binding domains; and
(d) isolating those combinations of first and second binding domains which
combine together to produce a target-binding dimer.
10 The first and second epitopes are adjacent such that a multimeric ligand is capable of
binding to both epitopes simultaneously. This provides the ligand with the advantages of
increased avidity if binding. Where the epitopes are the same, the increased avidity is
obtained by the presence of multiple copies of the epitope on the target, allowing at least
two copies to be simultaneously bound in order to obtain the increased avidity effect.
15
The binding domains may be associated by several methods, as well as the use of linkers.
For example, the binding domains may comprise cys residues, avidin and streptavidin
groups or other means for non-covalent attachment post-synthesis; those combinations
which bind to the target efficiently will be isolated. Alternatively, a linker may be present
20 between the first and second binding domains, which are expressed as a single
polypeptide from a single vector, which comprises the first binding domain, the linker
and a repertoire of second binding domains, for instance as described above.
In a preferred aspect, the first and second binding domains associate naturally when
25 bound to antigen; for example, V H and V K domains, when bound to adjacent epitopes, will
naturally associate in a three-way interaction to form a stable dimer. Such associated
proteins can be isolated in a target binding assay. An advantage of this procedure is that
only binding domains which bind to closely adjacent epitopes, in the correct
conformation, will associate and thus be isolated as a result of their increased avidity for
30 the target.
In an alternative embodiment of the above aspect of the second configuration of the
invention, at least one epitope binding domain comprises a non-immuno globulin 'protein
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scaffold' or 'protein skeleton' as herein defined. Suitable non-immunoglobulin protein
scaffolds include but are not limited to any of those selected from the group consisting of:
SpA, fibronectin, GroEL and other chaperones, lipocallin, CCTLA4 and affibodies, as set
forth above.
According to the above aspect of the second configuration of the invention,
advantageously, the epitope binding domains are attached to a 'protein skeleton'.
Advantageously, a protein skeleton according to the invention is an immunoglobulin
skeleton.
According to the present invention, the term 'immunoglobulin skeleton' refers to a
protein which comprises at least one immunoglobulin fold and which acts as a nucleus for
one or more epitope binding domains, as defined herein.
Preferred immunoglobulin skeletons as herein defined includes any one or more of those
selected from the following: an immunoglobulin molecule comprising at least (i) the CL
(kappa or lambda subclass) domain of an antibody; or (ii) the CHI domain of an antibody
heavy chain; an immunoglobulin molecule comprising the CHI and CH2 domains of an
antibody heavy chain; an immunoglobulin molecule comprising the CHI, CH2 and CHS
domains of an antibody heavy chain; or any of the subset (ii) in conjunction with the CL
(kappa or lambda subclass) domain of an antibody. A hinge region domain may also be
included. Such combinations of domains may, for example, mimic natural antibodies,
such as IgG or IgM, or fragments thereof, such as Fv, scFv, Fab or F(ab') 2 molecules.
Those skilled in the art will be aware that this list is not intended to be exhaustive.
Linking of the skeleton to the epitope binding domains, as herein defined may be
achieved at the polypeptide level, that is after expression of the nucleic acid encoding the
skeleton and/or the epitope binding domains. Alternatively, the linking step may be
performed at the nucleic acid level. Methods of linking a protein skeleton according to the
present invention, to the one or more epitope binding domains include the use of protein
chemistry and/or molecular biology techniques which will be familiar to those skilled in
the art and are described herein.
WO 2004/003019 PCT/GB2003/002804
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Advantageously, the closed conformation multispecific ligand may comprise a first
domain capable of binding a target molecule, and a second domain capable of binding a
molecule or group which extends the half-life of the ligand. For example, the molecule or
group may be a bulky agent, such as HSA or a cell matrix protein. As used herein, the
5 phrase "molecule or group which extends the half-life of a ligand" refers to a molecule or
chemical group which, when bound by a dual-specific ligand as described herein
increases the in vivo half-life of such dual specific ligand when administered to an
animal, relative to a ligand that does not bind that molecule or group. Examples of
molecules or groups that extend the half-life of a ligand are described hereinbelow. In a
10 preferred embodiment, the closed conformation multispecific ligand may be capable of
binding the target molecule only on displacement of the half-life enhancing molecule or
group. Thus, for example, a closed conformation multispecific ligand is maintained in
circulation in the bloodstream of a subject by a bulky molecule such as HSA. When a
target molecule is encountered, competition between the binding domains of the closed
15 conformation multispecific ligand results in displacement of the HSA and binding of the
target.
Ligands according to any aspect of the present invention, as well as dAb monomers
useful in constructing such ligands, may advantageously dissociate from their cognate
20 target(s) with a Kd of 300nM to 5pM (ie, 3 x 10"" 7 to 5 x 10~ 12 M), preferably 50nM
to20pM, or 5nM to 200pM or InM to lOOpM, 1 x 1(T 7 M or less, 1 x 10" 8 M or less, 1 x
10~ 9 M or less, 1 x 10" 10 M or less, 1 x 10" 11 M or less; and/or a Koff rate constant of 5 x
10' 1 to 1 x 10" 7 S'\ preferably 1 x 10" 2 to 1 x 10~ 6 S" 1 , or 5 x 10' 3 to 1 x 10* 5 S'\ or 5 x 10" 1
S" 1 or less, or 1 x 10" 2 S" 1 or less, or 1 x 10* 3 S" 1 or less, or 1 x 10~ 4 S" 1 or less, or 1 x 10" 5 S~ l
25 or less, or 1 x 1CT 6 S" 1 or less as determined by surface plasmon resonance. The Kd rate
constand is defined as Kcff/K^.
In particular the invention provides an anti-TNFa dAb monomer (or dual specific ligand
comprising such a dAb), homodimer, heterodimer or homotrimer ligand, wherein each
30 dAb binds TNFoc. The ligand binds to TNFcc with a Kd of 300nM to 5pM (ie, 3 x 1(T 7 to
5 x 10" 12 M), preferably 50nM to 20pM, more preferably 5nM to 200pM and most
preferably InM to lOOpM; expressed in an alternative manner, the Kd is 1 x 10" 7 M or
less, preferably 1x10" M or less, more preferably 1 x 10 M or less, advantageously 1 x
WO 2004/003019 PCT/GB2003/002804
30
10" 10 M or less and most preferably 1 x 10' 11 M or less; and/or a Koff rate constant of 5 x
10" 1 to 1 x 10" 7 S~\ preferably 1 x 10~ 2 to 1 x 10~ 6 S" 1 , more preferably 5 x 10~ 3 to 1 x 10" 5
S" 1 , for example 5 x 10" 1 S" 1 or less, preferably 1 x 10" 2 S" 1 or less, more preferably 1x10"
3 S" 1 or less, advantageously 1 x 10~ 4 S" 1 or less, further advantageously 1 x 10" 5 S* 1 or less,
5 and most preferably 1x10" S" or less, as determined by surface plasmon resonance.
Preferably, the ligand neutralises TNFoc in a standard L929 assay with an ND50 of
500nM to 50pM, preferably or lOOnM to 50pM, advantageously lOnM to lOOpM, more
preferably InM to lOOpM; for example 50nM or less, preferably 5nM or less,
10 advantageously 500pM or less, more preferably 200pM or less and most preferably
lOOpM or less.
Preferably, the ligand inhibits binding of TNF alpha to TNF alpha Receptor I (p55
receptor) with an IC50 of 500nM to 50pM, preferably lOOnM to 50pM, more preferably
15 lOnM to lOOpM, advantageously InM to lOOpM; for example 50nM or less, preferably
5nM or less, more preferably 500pM or less, advantageously 200pM or less, and most
preferably lOOpM or less. Preferably, the TNFoc is Human TNF a.
Furthermore, the invention provides a an anti-TNF Receptor I dAb monomer, or dual
20 specific ligand comprising such a dAb, that binds to TNF Receptor I with a IQ of 300nM
to 5pM (ie, 3 x 10' 7 to 5 x 10' 12 M), preferably 50nM to20pM, more preferably 5nM to
200pM and most preferably InM to lOOpM, for example 1 x 10" 7 M or less, preferably 1
x 10" 8 M or less, more preferably 1 x 10" 9 M or less, advantageously 1 x 10" 10 M or less
and most preferably 1 x 10" 11 M or less; and/or a Koff rate constant of 5 x 10" 1 to 1 x 10" 7
25 S'\ preferably 1 x 10" 2 to 1 x 10" 6 S"\ more preferably 5 x 10" 3 to 1 x 10' 5 S" 1 , for
example 5 x 10" 1 S" 1 or less, preferably 1 x 10" 2 S" 1 or less, advantageously 1 x 10" 3 S" 1 or
less, more preferably 1 x lO^S" 1 or less, still more preferably 1 x 10" 5 S" 1 or less, and most
preferably 1x10 S or less as determined by surface plasmon resonance.
30 Preferably, the dAb monomeror ligand neutralises TNFoc in a standard assay (eg, the
L929 or HeLa assays described herein) with an ND50 of 500nM to 50pM, preferably
lOOnM to 50pM, more preferably lOnM to lOOpM, advantageously InM to lOOpM; for
WO 2004/003019 PCT/GB2003/002804
31
example 50nM or less, preferably 5nM or less, more preferably 500pM or less,
advantageously 200pM or less, and most preferably lOOpM or less.
Preferably, the dAb monomer or ligand inhibits binding of TNF alpha to TNF alpha
5 Receptor I (p55 receptor) with an IC50 of 500nM to 50pM, preferably lOOnM to 50pM,
more preferably lOnM to lOOpM, advantageously InM to lOOpM; for example 50nM or
less, preferably 5nM or less, more preferably 500pM or less, advantageously 200pM or
less, and most preferably lOOpM or less. Preferably, the TNF Receptor I target is Human
TNFcc.
10
Furthermore, the invention provides a dAb monomer(or dual specific ligand comprising
such a dAb) that binds to serum albumin (SA) with a Kid of InM to 500/xM (ie, x 10~ 9 to
5x10"), preferably lOOnM to 10/xM. Preferably, for a dual specific ligand comprising a
first anti-SA dAb and a second dAb to another target, the affinity (eg and/or Ko ff as
15 measured by surface plasmon resonance, eg using BiaCore) of the second dAb for its
target is from 1 to 100000 times (preferably 100 to 100000, more preferably 1000 to
100000, or 10000 to 100000 times) the affinity of the first dAb for SA. For example, the
first dAb binds SA with an affinity of approximately 10/xM, while the second dAb binds
its target with an affinity of lOOpM. Preferably, the serum albumin is human serum
20 albumin (HSA).
In one embodiment, the first dAb (or a dAb monomer) binds SA (eg, HSA) with a K4 of
approximately 50, preferably 70, and more preferably 100, 150 or 200 nM.
25 The invention moreover provides dimers, trimers and polymers of the aforementioned
dAb monomers, in accordance with the foregoing aspect of the present invention.
Ligands according to the invention, including dAb monomers, dimers and trimers, can be
linked to an antibody Fc region, comprising one or both of Cr2 and Ch3 domains, and
30 optionally a hinge region. For example, vectors encoding ligands linked as a single
nucleotide sequence to an Fc region may be used to prepare such polypeptides.
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In a further aspect of the second configuration of the invention, the present invention
provides one or more nucleic acid molecules encoding at least a multispecific ligand as
herein defined. In one embodiment, the ligand is a closed conformation ligand. In
another embodiment, it is an open conformation ligand. The multispecific ligand may be
5 encoded on a single nucleic acid molecule; alternatively, each epitope binding domain
may be encoded by a separate nucleic acid molecule. Where the ligand is encoded by a
single nucleic acid molecule, the domains may be expressed as a fusion polypeptide, or
may be separately expressed and subsequently linked together, for example using
chemical linking agents. Ligands expressed from separate nucleic acids will be linked
10 together by appropriate means.
The nucleic acid may further encode a signal sequence for export of the polypeptides
from a host cell upon expression and may be fused with a surface component of a
filamentous bacteriophage particle (or other component of a selection display system)
15 upon expression. Leader sequences, which may be used in bacterial expresion and/or
phage or phagemid display, include pelB, stll, ompA, phoA, bla and pelA.
In a further aspect of the second configuration of the invention the present invention
provides a vector comprising nucleic acid according to the present invention.
20
In a yet further aspect, the present invention provides a host cell transfected with a vector
according to the present invention.
Expression from such a vector may be configured to produce, for example on the surface
25 of a bacteriophage particle, epitope binding domains for selection. This allows selection
of displayed domains and thus selection of 'multispecific ligands' using the method of the
present invention.
In a preferred embodiment of the second configuration of the invention, the epitope
30 binding domains are immunoglobulin variable regions and are selected from single
domain V gene repertoires. Generally the repertoire of single antibody domains is
displayed on the surface of filamentous bacteriophage. In a preferred embodiment each
single antibody domain is selected by binding of a phage repertoire to antigen.
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The present invention further provides a kit comprising at least a multispecific ligand
according to the present invention, which may be an open conformation or closed
conformation ligand. Kits according to the invention may be, for example, diagnostic
kits, therapeutic kits, kits for the detection of chemical or biological species, and the like.
In a further aspect still of the second configuration of the invention, the present invention
provides a homogenous immunoassay using a ligand according to the present invention.
In a further aspect still of the second configuration of the invention, the present invention
provides a composition comprising a closed conformation multispecific ligand, obtainable
by a method of the present invention, and a pharmaceutically acceptable carrier, diluent or
excipient.
Moreover, the present invention provides a method for the treatment of disease using a
"closed conformation multispecific ligand 5 or a composition according to the present
invention.
In a preferred embodiment of the invention the disease is cancer or an inflammatory
disease, eg rheumatoid arthritis, asthma or Crohn's disease.
In a further aspect of the second configuration of the invention, the present invention
provides a method for the diagnosis, including diagnosis of disease using a closed
conformation multispecific ligand, or a composition according to the present invention.
Thus in general the binding of an analyte to a closed conformation multispecific ligand
may be exploited to displace an agent, which leads to the generation of a signal on
displacement. For example, binding of analyte (second antigen) could displace an
enzyme (first antigen) bound to the antibody providing the basis for an immunoassay,
especially if the enzyme were held to the antibody through its active site.
Thus in a final aspect of the second configuration, the present invention provides a
method for detecting the presence of a target molecule, comprising:
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(a) providing a closed conformation multispecific ligand bound to an agent, said ligand
being specific for the target molecule and the agent, wherein the agent which is bound by
the ligand leads to the generation of a detectable signal on displacement from the ligand;
(b) exposing the closed conformation multispecific ligand to the target molecule; and
(c) detecting the signal generated as a result of the displacement of the agent.
According to the above aspect of the second configuration of the invention,
advantageously, the agent is an enzyme, which is inactive when bound by the closed
conformation multi-specific ligand. Alternatively, the agent may be any one or more
selected from the group consisting of the following: the substrate for an enzyme, and a
fluorescent, luminescent or chromogenic molecule which is inactive or quenched when
bound by the ligand.
Sequences similar or homologous (e.g., at least about 70% sequence identity) to the
sequences disclosed herein are also part of the invention. In some embodiments, the
sequence identity at the amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the sequence
identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or higher. Alternatively, substantial identity exists when the nucleic acid
segments will hybridize under selective hybridization conditions (e.g., very high
stringency hybridization conditions), to the complement of the strand. The nucleic acids
may be present in whole cells, in a cell lysate, or in a partially purified or substantially
pure form.
Calculations of "homology" or "sequence identity" or "similarity" between two
sequences (the terms are used interchangeably herein) are performed as follows. The
sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in
one or both of a first and a second amino acid or nucleic acid sequence for optimal
alignment and non-homologous sequences can be disregarded for comparison purposes).
In a preferred embodiment, the length of a reference sequence aligned for comparison
purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more
preferably at least 60%, and even more preferably at least 70%, 80%), 90%, 100% of the
length of the reference sequence. The amino acid residues or nucleotides at
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corresponding amino acid positions or nucleotide positions are then compared. When a
position in the first sequence is occupied by the same amino acid residue or nucleotide as
the corresponding position in the second sequence, then the molecules are identical at that
position (as used herein amino acid or nucleic acid "homology" is equivalent to amino
acid or nucleic acid "identity"). The percent identity between the two sequences is a
function of the number of identical positions shared by the sequences, taking into account
the number of gaps, and the length of each gap, which need to be introduced for optimal
alignment of the two sequences.
Advantageously, the BLAST algorithm (version 2.0) is employed for sequence alignment,
with parameters set to default values. The BLAST algorithm is described in detail at the
world wide web site ("www") of the National Center for Biotechnology Information
(".ncbi") of the National Institutes of Health ("nih") of the U.S. government (".gov"), in
the "/Blast/" directory, in the "blast_help.html" file. The search parameters are defined as
follows, and are advantageously set to the defined default parameters.
BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm employed
by the programs blastp, blastn, blastx, tblastn, and tblastx; these programs ascribe
significance to their findings using the statistical methods of Karlin and Altschul, 1990,
Proc. Natl. Acad. Sci. USA 87(6):2264-8 (see the "blast_help.html" file, as described
above) with a few enhancements. The BLAST programs were tailored for sequence
similarity searching, for example to identify homologues to a query sequence. The
programs are not generally useful for motif-style searching. For a discussion of basic
issues in similarity searching of sequence databases, see Altschul et al. (1994).
The five BLAST programs available at the National Center for Biotechnology
Information web site perform the following tasks:
"blastp" compares an amino acid query sequence against a protein sequence database;
"blastn" compares a nucleotide query sequence against a nucleotide sequence database;
"blastx" compares the six-frame conceptual translation products of a nucleotide query
sequence (both strands) against a protein sequence database;
"tblastn" compares a protein query sequence against a nucleotide sequence database
dynamically translated in all six reading frames (both strands).
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"tblastx" compares the six-frame translations of a nucleotide query sequence against the
six-frame translations of a nucleotide sequence database.
BLAST uses the following search parameters:
HISTOGRAM Display a histogram of scores for each search; default is yes. (See
5 parameter H in the BLAST Manual).
DESCRIPTIONS Restricts the number of short descriptions of matching sequences
reported to the number specified; default limit is 100 descriptions. (See parameter V in
the manual page). See also EXPECT and CUTOFF.
ALIGNMENTS Restricts database sequences to the number specified for which high-
10 scoring segment pairs (HSPs) are reported; the default limit is 50. If more database
sequences than this happen to satisfy the statistical significance threshold for reporting
(see EXPECT and CUTOFF below), only the matches ascribed the greatest statistical
significance are reported. (See parameter B in the BLAST Manual).
EXPECT The statistical significance threshold for reporting matches against database
15 sequences; the default value is 10, such that 10 matches are expected to be found merely
by chance, according to the stochastic model of Karlin and Altschul (1990). If the
statistical significance ascribed to a match is greater than the EXPECT threshold, the
match will not be reported. Lower EXPECT thresholds are more stringent, leading to
fewer chance matches being reported. Fractional values are acceptable. (See parameter E
20 in the BLAST Manual).
CUTOFF Cutoff score for reporting high-scoring segment pairs. The default value is
calculated from the EXPECT value (see above). HSPs are reported for a database
sequence only if the statistical significance ascribed to them is at least as high as would be
ascribed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF
25 values are more stringent, leading to fewer chance matches being reported. (See
parameter S in the BLAST Manual). Typically, significance thresholds can be more
intuitively managed using EXPECT.
MATRIX Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and
TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992, Proc. Natl.
30 Aacad. Sci. USA 89(22): 10915-9). The valid alternative choices include: PAM40,
PAM120, PAM250 and IDENTITY. No alternate scoring matrices are available for
BLASTN; specifying the MATRIX directive in BLASTN requests returns an error
response.
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STRAND Restrict a TBLASTN search to just the top or bottom strand of the database
sequences; or restrict a BLASTN, BLASTX or TBLASTX search to just reading frames
on the top or bottom strand of the query sequence.
FILTER Mask off segments of the query sequence that have low compositional
5 complexity, as determined by the SEG program of Wootton & Federhen (1993)
Computers and Chemistry 17:149-163, or segments consisting of short-periodicity
internal repeats, as determined by the XNU program of Claverie & States, 1993,
Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST program of
Tatusov and Lipman (see the world wide web site of the NCBI). Filtering can eliminate
10 statistically significant but biologically uninteresting reports from the blast output (e.g.,
hits against common acidic-, basic- or pro line-rich regions), leaving the more biologically
interesting regions of the query sequence available for specific matching against database
sequences.
15 Low complexity sequence found by a filter program is substituted using the letter "N" in
nucleotide sequence (e.g., "N" repeated 13 times) and the letter "X" in protein sequences
(e.g., "X" repeated 9 times).
Filtering is only applied to the query sequence (or its translation products), not to
20 database sequences. Default filtering is DUST for BLASTN, SEG for other programs.
It is not unusual for nothing at all to be masked by SEG, XNU, or both, when applied to
sequences in SWISS-PROT, so filtering should not be expected to always yield an effect.
Furthermore, in some cases, sequences are masked in their entirety, indicating that the
statistical significance of any matches reported against the unfiltered query sequence
25 should be suspect.
NCBI-gi Causes NCBI gi identifiers to be shown in the output, in addition to the
accession and/or locus name.
30 Most preferably, sequence comparisons are conducted using the simple BLAST search
algorithm provided at the NCBI world wide web site described above, in the "/BLAST"
directory.
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Brief Description of the Figures
Figure 1 shows the diversification of V H /HSA at positions H50, H52, H52a, H53,
H55, H56, H58, H95, H96, H97, H98 (DVT or NNK encoded
respectively) which are in the antigen binding site of V H HSA. The
sequence of V K is diversified at positions L50, L53.
Figure 2 shows Library 1 : Germline V K /DVT Vh,
Library 2: Germline V K /NNK V H ,
Library 3: Germline V H /DVT V K
Library 4: Germline V H /NNK V K
In phage display/ScFv format. These libraries were pre-selected for
binding to generic ligands protein A and protein L so that the majority of
the clones and selected libraries are functional. Libraries were selected on
HSA (first round) and /3-gal (second round) or HSA /3-gal selection or on
/?-gal (first round) and HSA (second round) /3-gal HSA selection. Soluble
scFv from these clones of PCR are amplified in the sequence. One clone
encoding a dual specific antibody K8 was chosen for further work.
Figure 3 shows an alignment of Vh chains and V K chains.
Figure 4 shows the characterisation of the binding properties of the K8 antibody,
the binding properties of the K8 antibody characterised by monoclonal
phage ELISA, the dual specific K8 antibody was found to bind HSA and
jS-gal and displayed on the surface of the phage with absorbant signals
greater than 1.0. No cross reactivity with other proteins was detected.
Figure 5 shows soluble scFv ELISA performed using known concentrations of the
K8 antibody fragment. A 96-well plate was coated with 100/ig of HSA,
BSA and /3-gal at lOjttg/ml and 100/xg/ml of Protein A at 1/ig/ml
concentration. 50 fig of the serial dilutions of the K8 scFv was applied and
the bound antibody fragments were detected with Protein L-HRP. ELISA
results confirm the dual specific nature of the K8 antibody.
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Figure 6
shows the binding characteristics of the clone OV^/dummy Vh analysed
using soluble scFv ELISA. Production of the soluble scFv fragments was
induced by IPTG as described by Harrison et al, Methods Enzymol.
1996;267:83-109 and the supernatant containing scFv assayed directly.
Soluble scFv ELISA is performed as described in example 1 and the bound
scFvs were detected with Protein L-HRP. The ELISA results revealed that
this clone was still able to bind /3-gal, whereas binding BS A was abolished.
10 Figure 7
shows the sequence of variable domain vectors 1 and 2.
Figure 8
is a map of the Ch vector used to construct a Vh1/Vh2 multipsecific
ligand.
15 Figure 9
is a map of the V K vector used to construct a V K 1 /V K 2 multispecific ligand.
Figure 10 TNF receptor assay comparing TAR1-5 dimer 4, TAR1-5-19 dimer 4 and
TAR1-5-19 monomer.
20 Figure 1 1
TNF receptor assay comparing TAR1-5 dimers 1-6. All dimers have been
FPLC purified and the results for the optimal dimeric species are shown.
25
Figure 12
TNF receptor assay of TAR1-5 19 homodimers in different formats: dAb-
linker-dAb format with 3U, 5U or 7U linker, Fab format and cysteine
hinge linker format.
30
Figure 13
4
Dummy VH sequence for library 1 . The sequence of the VH framework
based on germline sequence DP47 - JH4b. Positions where NNK
randomisation (N=A or T or C or G nucleotides; K = G or T nucleotides)
has been incorporated into library 1 are indicated in bold underlined text.
Figure 14
Dummy VH sequence for library 2. The sequence of the VH framework
based on germline sequence DP47 - JH4b. Positions where NNK
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randomisation (N=A or T or C or G nucleotides; K = G or T nucleotides)
has been incorporated into library 2 are indicated in bold underlined text.
Dummy Vk sequence for library 3. The sequence of the V/c framework
based on germline sequence DPk9 - J k1. Positions where NNK
randomisation (N=A or T or C or G nucleotides; K = G or T nucleotides)
has been incorporated into library 3 are indicated in bold underlined text.
Nucleotide and amino acid sequence of anti MSA dAbs MSA 16 and MSA
26.
Inhibition biacore of MSA 16 and 26. Purified dAbs MSA16 and MSA26
were analysed by inhibition biacore to determine Kd. Briefly, the dAbs
were tested to determine the concentration of dAb required to achieve
200RUs of response on a biacore CM5 chip coated with a high density of
MSA. Once the required concentrations of dAb had been determined,
MSA antigen at a range of concentrations around the expected Kd was
premixed with the dAb and incubated overnight. Binding to the MSA
coated biacore chip of dAb in each of the premixes was then measured at a
high flow-rate of 30 /d/minute.
Serum levels of MSA16 following injection. Serum half life of the dAb
MSA16 was determined in mouse. MSA16 was dosed as single i.v.
injections at approx 1.5mg/kg into CD1 mice. Modelling with a 2
compartment model showed MSA16 had a Mi 2a of 0.98hr, a 1 1/2)8 of
36.5hr and an AUC of 913hr.mg/ml. MSA16 had a considerably
lengthened half life compared with HEL4 (an anti-hen egg white lysozyme
dAb) which had a tl/2aof 0.06hr and atl/2/3 of 0.34hr.
ELIS A (a) and TNF receptor assay (c) showing inhibition of TNP binding
with a Fab-like fragment comprising MSA26Ck and TAR1-5-19CH.
Addition of MSA with the Fab-like fragment reduces the level of
inhibition. An ELIS A plate coated with l/ig/ml TNFa was probed with
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dual specific Vk C h and V/c C/c Fab like fragment and also with a control
TNFa binding dAb at a concentration calculated to give a similar signal on
the ELISA. Both the dual specific and control dAb were used to probe the
ELISA plate in the presence and in the absence of 2mg/ml MSA. The
signal in the dual specific well was reduced by more than 50% but the
signal in the dAb well was not reduced at all (see figure 19a). The same
dual specific protein was also put into the receptor assay with and without
MSA and competition by MSA was also shown (see figure 19c). This
demonstrates that binding of MSA to the dual specific is competitive with
binding to TNFa.
Figure 20 TNF receptor assay showing inhibiton of TNF binding with a disulphide
bonded heterodimer of TAR1-5-19 dAb and MSA16 dAb. Addition of
MSA with the dimer reduces the level of inhibiton in a dose dependant
manner. The TNF receptor assay (figure 19 (b)) was conducted in the
presence of a constant concentration of heterodimer (18nM) and a dilution
series of MSA and HSA. The presence of HSA at a range of
concentrations (up to 2 mg/ml) did not cause a reduction in the ability of
the dimer to inhibit TNFa . However, the addition of MSA caused a dose
dependant reduction in the ability of the dimer to inhibit TNFa (figure
19a).This demonstrates that MSA and TNFa compete for binding to the
cys bonded TARl-5^19, MSA16 dimer. MSA and HSA alone did not
have an effect on the TNF binding level in the assay.
Detailed Description of the Invention
Definitions
Complementary Two immunoglobulin domains are "complementary 55 where they
belong to families of structures which form cognate pairs or groups or are derived from
such families and retain this feature. For example, a V H domain and a V L domain of an
antibody are complementary; two Vh domains are not complementary, and two V L
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domains are not complementary. Complementary domains may be found in other
members of the immunoglobulin superfamily, such as the V a and V p (or y and 5) domains
of the T-cell receptor. In the context of the second configuration of the present invention,
non-complementary domains do not bind a target molecule cooperatively, but act
5 independently on different target epitopes which may be on the same or different
molecules. Domains which are artificial, such as domains based on protein scaffolds
which do not bind epitopes unless engineered to do so, are non-complementary.
Likewise, two domains based on (for example) an immunoglobulin domain and a
fibronectin domain are not complementary.
10
Immunoglobulin This refers to a family of polypeptides which retain the
immunoglobulin fold characteristic of antibody molecules, which contains two 0 sheets
and, usually, a conserved disulphide bond. Members of the immunoglobulin superfamily
are involved in many aspects of cellular and non-cellular interactions in vivo, including
15 widespread roles in the immune system (for example, antibodies, T-cell receptor
molecules and the like), involvement in cell adhesion (for example the ICAM molecules)
and intracellular signalling (for example, receptor molecules, such as the PDGF receptor).
The present invention is applicable to all immunoglobulin superfamily molecules which
possess binding domains. Preferably, the present invention relates to antibodies.
20
Combining Variable domains according to the invention are combined to form a group
of domains; for example, complementary domains may be combined, such as Vl domains
being combined with V H domains. Non-complementary domains may also be combined.
Domains may be combined in a number of ways, involving linkage of the domains by
25 covalent or non-covalent means.
Domain A domain is a folded protein structure which retains its tertiary structure
independently of the rest of the protein. Generally, domains are responsible for discrete
functional properties of proteins, and in many cases may be added, removed or
30 transferred to other proteins without loss of function of the remainder of the protein
and/or of the domain. By single antibody variable domain is meant a folded polypeptide
domain comprising sequences characteristic of antibody variable domains. It therefore
includes complete antibody variable domains and modified variable domains, for example
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in which one or more loops have been replaced by sequences which are not characteristic
of antibody variable domains, or antibody variable domains which have been truncated or
comprise N- or C-terminal extensions, as well as folded fragments of variable domains
which retain at least in part the binding activity and specificity of the full-length domain.
5
Repertoire A collection of diverse variants, for example polypeptide variants which
differ in their primary sequence. A library used in the present invention will encompass a
repertoire of polypeptides comprising at least 1000 members.
10 Library The term library refers to a mixture of heterogeneous polypeptides or
nucleic acids. The library is composed of members, each of which have a single
polypeptide or nucleic acid sequence. To this extent, library is synonymous with
repertoire. Sequence differences between library members are responsible for the
diversity present in the library. The library may take the form of a simple mixture of
15 polypeptides or nucleic acids, or may be in the form of organisms or cells, for example
bacteria, viruses, animal or plant cells and the like, transformed with a library of nucleic
acids. Preferably, each individual organism or cell contains only one or a limited number
of library members. Advantageously, the nucleic acids are incorporated into expression
vectors, in order to allow expression of the polypeptides encoded by the nucleic acids. In
20 a preferred aspect, therefore, a library may take the form of a population of host
organisms, each organism containing one or more copies of an expression vector
containing a single member of the library in nucleic acid form which can be expressed to
produce its corresponding polypeptide member. Thus, the population of host organisms
has the potential to encode a large repertoire of genetically diverse polypeptide variants.
25
A c closed conformation multi-specific ligand 5 describes a multi-specific ligand as
herein defined comprising at least two epitope binding domains as herein defined. The
term 'closed conformation' (multi-specific ligand) means that the epitope binding
domains of the ligand are arranged such that epitope binding by one epitope binding
30 domain competes with epitope binding by another epitope binding domain. That is,
cognate epitopes may be bound by each epitope binding domain individually but not
simultaneosuly. The closed conformation of the ligand can be achieved using methods
herein described.
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Antibody An antibody (for example IgG, IgM, IgA, IgD or IgE) or fragment (such
as a Fab , F(ab') 2 , Fv, disulphide linked Fv 5 scFv ? closed conformation multispecific
antibody, disulphide-linked scFv, diabody) whether derived from any species naturally
producing an antibody, or created by recombinant DNA technology; whether isolated
5 from serum, B-cells, hybridomas, transfectomas, yeast or bacteria).
Dual-specific ligand A ligand comprising a first immunoglobulin single variable domain
and a second immunoglobulin single variable domain as herein defined, wherein the
variable regions are capable of binding to two different antigens or two epitopes on the
10 same antigen which are not normally bound by a monospecific immunoglobulin. For
example, the two epitopes may be on the same hapten, but are not the same epitope or
sufficiently adjacent to be bound by a monospecific ligand. The dual specific ligands
according to the invention are composed of variable domains which have different
specificities, and do not contain mutually complementary variable domain pairs which
15 have the same specificity.
Antigen A molecule that is bound by a ligand according to the present invention.
Typically, antigens are bound by antibody ligands and are capable of raising an antibody
response in vivo. It may be a polypeptide, protein, nucleic acid or other molecule.
20 Generally, the dual specific ligands according to the invention are selected for target
specificity against a particular antigen. In the case of conventional antibodies and
fragments thereof, the antibody binding site defined by the variable loops (LI, L2, L3 and
HI, H2, H3) is capable of binding to the antigen.
25 Epitope A unit of structure conventionally bound by an immunoglobulin Vr/Vl
pair. Epitopes define the minimum binding site for an antibody, and thus represent the
target of specificity of an antibody. In the case of a single domain antibody, an epitope
represents the unit of structure bound by a variable domain in isolation.
30 Generic ligand A ligand that binds to all members of a repertoire. Generally, not bound
through the antigen binding site as defined above. Non-limiting examples include protein
A, protein L and protein G.
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Selecting Derived by screening, or derived by a Darwinian selection process, in
which binding interactions are made between a domain and the antigen or epitope or
between an antibody and an antigen or epitope. Thus a first variable domain may be
selected for binding to an antigen or epitope in the presence or in the absence of a
5 complementary variable domain.
Universal framework A single antibody framework sequence corresponding to
the regions of an antibody conserved in sequence as defined by Kabat ("Sequences of
Proteins of Immunological Interest", US Department of Health and Human Services) or
10 corresponding to the human germline immunoglobulin repertoire or structure as defined
by Chothia and Lesk, (1987) J. Mol. Biol. 196:910-917. The invention provides for the
use of a single framework, or a set of such frameworks, which has been found to permit
the derivation of virtually any binding specificity though variation in the hypervariable
regions alone.
15
Half-life The time taken for the serum concentration of the ligand to reduce by 50%,
in vivo, for example due to degradation of the ligand and/or clearance or sequestration of
the ligand by natural mechanisms. The ligands of the invention are stabilised in vivo and
their half-life increased by binding to molecules which resist degradation and/or clearance
20 or sequestration. Typically, such molecules are naturally occurring proteins which
themselves have a long half-life in vivo. The half-life of a ligand is increased if its
functional activity persists, in vivo, for a longer period than a similar ligand which is not
specific for the half-life increasing molecule. Thus, a ligand specific for HSA and a target
molecule is compared with the same ligand wherein the specificity for HSA is not
25 present, that it does not bind HSA but binds another molecule. For example, it may bind
a second epitope on the target molecule. Typically, the half life is increased by 1 0%,
20%, 30%, 40%, 50% or more. Increases in the range of 2x, 3x, 4x, 5x, lOx, 20x, 30x,
40x, 5 Ox or more of the half life are possible. Alternatively, or in addition, increases in
the range of up to 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx, 150x of the half life are
30 possible.
Homogeneous immunoassay An immunoassay in which analyte is detected
without need for a step of separating bound and un-bound reagents.
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Substantially identical (or "substantially homologous") A first amino acid or
nucleotide sequence that contains a sufficient number of identical or equivalent (e.g. 9 with
a similar side chain, e.g., conserved amino acid substitutions) amino acid residues or
5 nucleotides to a second amino acid or nucleotide sequence such that the first and second
amino acid or nucleotide sequences have similar activities. In the case of antibodies, the
second antibody has the same binding specificity and has at least 50% of the affinity of
the same.
10 As used herein, the terms "low stringency " "medium stringency/' "high stringency/ 5
or "very high stringency conditions" describe conditions for nucleic acid hybridization
and washing. Guidance for performing hybridization reactions can be found in Current
Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is
incorporated herein by reference in its entirety. Aqueous and nonaqueous methods are
15 described in that reference and either can be used. Specific hybridization conditions
referred to herein are as follows: (1) low stringency hybridization conditions in 6X
sodium chloride/sodium citrate (SSC) at about 45 °C, followed by two washes in 0.2X
SSC, 0.1% SDS at least at 50°C (the temperature of the washes can be increased to 55°C
for low stringency conditions); (2) medium stringency hybridization conditions in 6X
20 SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; (3)
high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or
more washes in 0.2X SSC, 0.1% SDS at 65°C; and preferably (4) very high stringency
hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one
or more washes at 0.2X SSC, 1% SDS at 65°C. Very high stringency conditions (4) are
25 the preferred conditions and the ones that should be used unless otherwise specified.
Detailed Description of the Invention
30 Unless defined otherwise, all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture,
molecular genetics, nucleic acid chemistry, hybridisation techniques and biochemistry).
Standard techniques are used for molecular, genetic and biochemical methods (see
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generally, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed. (1989)
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al, Short
Protocols in Molecular Biology (1999) 4 th Ed, John Wiley & Sons, Inc. which are
incorporated herein by reference) and chemical methods.
Preparation of immunoglobulin based multi-specific Iigands
Dual specific Iigands according to the invention, whether open or closed in conformation
according to the desired configuration of the invention, may be prepared according to
previously established techniques, used in the field of antibody engineering, for the
preparation of scFv, "phage" antibodies and other engineered antibody molecules.
Techniques for the preparation of antibodies, and in particular bispecific antibodies, are
for example described in the following reviews and the references cited therein: Winter &
Milstein, (1991) Nature 349:293-299; Plueckthun (1992) Immunological Reviews
130:151-188; Wright et al, (1992) Crti. Rev. Immunol.l2:125-168; Holliger, P. &
Winter, G. (1993) Curr. Op. Biotechn. 4, 446-449; Carter, et al. (1995) J. Hematother. 4,
463-470; Chester, K.A. & Hawkins, RE. (1995) Trends Biotechn. 13, 294-300;
Hoogenboom, H.R. (1997) Nature Biotechnol. 15, 125-126;' Fearon, D. (1997) Nature
Biotechnol. 15, 618-619; Pliickthun, A. & Pack, P. (1997) Immunotechnology 3, 83-105;
Carter, P. & Merchant, A.M. (1997) Curr. Opin. Biotechnol. 8, 449-454; Holliger, P. &
Winter, G. (1997) Cancer Immunol. Immunother. 45,128-130.
The invention provides for the selection of variable domains against two different
antigens or epitopes, and subsequent combination of the variable domains.
The techniques employed for selection of the variable domains employ libraries and
selection procedures which are known in the art. Natural libraries (Marks et al (1991) J.
Mol Biol., 222: 581; Vaughan et al. (1996) Nature Biotech., 14: 309) which use
rearranged V genes harvested from human B cells are well known to those skilled in the
art. Synthetic libraries (Hoogenboom & Winter (1992) /. Mol. Biol, 227: 381; Barbas et
al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457; Nissim et al. (1994) EMBO J., 13: 692;
Griffiths et al. (1994) EMBO J., 13: 3245; De Kruif et al. (1995) J. Mol. Biol, 248: 97)
are prepared by cloning immunoglobulin V genes, usually using PCR. Errors in the PCR
WO 2004/003019 PCT/GB2003/002804
48
process can lead to a high degree of randomisation. Vh and/or Vl libraries may be
selected against target antigens or epitopes separately, in which case single domain
binding is directly selected for, or together.
A preferred method for making a dual specific ligand according to the present invention
comprises using a selection system in which a repertoire of variable domains is selected
for binding to a first antigen or epitope and a repertoire of variable domains is selected for
binding to a second antigen or epitope. The selected variable first and second variable
domains are then combined and the dual-specific ligand selected for binding to both first
and second antigen or epitope. Closed conformation ligands are selected for binding both
first and second antigen or epitope in isolation but not simultaneously.
A. Library vector systems
A variety of selection systems are known in the art which are suitable for use in the
present invention. Examples of such systems are described below.
Bacteriophage lambda expression systems may be screened directly as bacteriophage
plaques or as colonies of lysogens, both as previously described (Huse et al (1989,1
Science, 246: 1275; Caton and Koprowski (1990) Proc. Natl. Acad. Set U.S.A., 87;
Mullinax et al. (1990) Proc. Natl Acad. Sci. U.S.A., 87: 8095; Persson et al. (1991) Proc.
Natl. Acad. Sci. U.S.A., 88: 2432) and are of use in the invention. Whilst such expression
systems can be used to screen up to 10 6 different members of a library, they are not really
suited to screening of larger numbers (greater than 10 6 members).
Of particular use in the construction of libraries are selection display systems, which
enable a nucleic acid to be linked to the polypeptide it expresses. As used herein, a
selection display system is a system that permits the selection, by suitable display means,
of the individual members of the library by binding the generic and/or target ligands.
Selection protocols for isolating desired members of large libraries are known in the art,
as typified by phage display techniques. Such systems, in which diverse peptide
sequences are displayed on the surface of filamentous bacteriophage (Scott and Smith
WO 2004/003019 PCT/GB2003/002804
49
(1990) Science, 249: 386), have proven useful for creating libraries of antibody
fragments (and the nucleotide sequences that encoding them) for the in vitro selection and
amplification of specific antibody fragments that bind a target antigen (McCafferty et al,
WO 92/01047). The nucleotide sequences encoding the V H and V L regions are linked to
5 gene fragments which encode leader signals that direct them to the periplasmic space of
E. coli and as a result the resultant antibody fragments are displayed on the surface of the
bacteriophage, typically as fusions to bacteriophage coat proteins (e.g., pill or pVTII).
Alternatively, antibody fragments are displayed externally on lambda phage capsids
(phagebodies). An advantage of phage-based display systems is that, because they are
10 biological systems, selected library members can be amplified simply by growing the
phage containing the selected library member in bacterial cells. Furthermore, since the
nucleotide sequence that encode the polypeptide library member is contained on a phage
or phagemid vector, sequencing, expression and subsequent genetic manipulation is
relatively straightforward.
15
Methods for the construction of bacteriophage antibody display libraries and lambda
phage expression libraries are well known in the art (McCafferty et al (1990) Nature,
348: 552; Kang et al (1991) Proc. Natl. Acad. Sci. U.S.A., 88: 4363; Clackson et al
(1991) Nature, 352: 624; Lowman et al (1991) Biochemistry, 30: 10832; Burton et al
20 (1991) Proc. Natl Acad. Sci U.S.A., 88: 10134; Hoogenboom et al (1991) Nucleic Acids
Res., 19: 4133; Chang et al (1991) J. Immunol, 147: 3610; Breitling et al (1991) Gene,
104: 147; Marks et al (1991) supra; Barbas et al (1992) supra; Hawkins and Winter
(1992) J. Immunol, 22: 867; Marks et al, 1992, J. Biol Chem., 267: 16007; Lemer et al
(1992) Science, 258: 1313, incorporated herein by reference).
25
One particularly advantageous approach has been the use of scFv phage-libraries (Huston
et al, 1988, Proc. Natl. Acad. Sci U.S.A., 85: 5879-5883; Chaudhary et al (1990) Proc.
Natl. Acad. Sci U.S.A., 87: 1066-1070; McCafferty et al (1990) supra; Clackson et al
(1991) Nature, 352: 624; Marks et al (1991) J. Mol Biol, 222: 581; Chiswell et al
30 (1992) Trends Biotech., 10: 80; Marks et al (1992) J. Biol Chem., 267). Various
embodiments of scFv libraries displayed on bacteriophage coat proteins have been
described. Refinements of phage display approaches are also known, for example as
WO 2004/003019
PCT/GB2003/002804
50
described in WO96/06213 and WO92/01047 (Medical Research Council et al) and
WO97/08320 (Morphosys), which are incorporated herein by reference.
Other systems for generating libraries of polypeptides involve the use of cell-free
5 enzymatic machinery for the in vitro synthesis of the library members. In one method,
RNA molecules are selected by alternate rounds of selection against a target ligand and
PCR amplification (Tuerk and Gold (1990) Science, 249: 505; Ellington and Szostak
(1990) Nature, 346: 818). A similar technique may be used to identify DNA sequences
which bind a predetermined human transcription factor (Thiesen and Bach (1990) Nucleic
10 Acids Res., 18: 3203; Beaudry and Joyce (1992) Science, 257: 635; WO92/05258 and
W092/14843). In a similar way, in vitro translation can be used to synthesise
polypeptides as a method for generating large libraries. These methods which generally
comprise stabilised polysome complexes, are described further in WO88/08453,
WO90/05785, WO90/07003, WO91/02076, WO91/05058, and WO92/02536. Alternative
15 display systems which are not phage-based, such as those disclosed in W095/22625 and
W095/1 1922 (Affymax) use the polysomes to display polypeptides for selection.
A still further category of techniques involves the selection of repertoires in artificial
compartments, which allow the linkage of a gene with its gene product. For example, a
20 selection system in which nucleic acids encoding desirable gene products may be selected
in microcapsules formed by water-in-oil emulsions is described in WO99/02671,
WO00/40712 and Tawfik & Griffiths (1998) Nature Biotechnol 16(7), 652-6. Genetic
elements encoding a gene product having a desired activity are compartmentalised into
microcapsules and then transcribed and/or translated to produce their respective gene
25 products (RNA or protein) within the microcapsules. Genetic elements which produce
gene product having desired activity are subsequently sorted. This approach selects gene
products of interest by detecting the desired activity by a variety of means.
30 B. Library Construction .
Libraries intended for selection, may be constructed using techniques known in the art,
for example as set forth above, or may be purchased from commercial sources. Libraries
WO 2004/003019 PCT/GB2003/002804
51
which are useful in the present invention are described, for example, in WO99/20749.
Once a vector system is chosen and one or more nucleic acid sequences encoding
polypeptides of interest are cloned into the library vector, one may generate diversity
within the cloned molecules by undertaking mutagenesis prior to expression;
5 alternatively, the encoded proteins may be expressed and selected, as described above,
before mutagenesis and additional rounds of selection are performed. Mutagenesis of
nucleic acid sequences encoding structurally optimised polypeptides is carried out by
standard molecular methods. Of particular use is the polymerase chain reaction, or PCR,
(Mullis and Faloona (1987) Methods Enzymol. , 155: 335, herein incorporated by
10 reference). PCR, which uses multiple cycles of DNA replication catalysed by a
thermostable, DNA-dependent DNA polymerase to amplify the target sequence of
interest, is well known in the art. The construction of various antibody libraries has been
discussed in Winter et ah (1994) Ann. Rev. Immunology 12, 433-55, and references cited
therein.
15
PCR is performed using template DNA (at least lfg; more usefully, 1-1000 ng) and at
least 25 pmol of oligonucleotide primers; it may be advantageous to use a larger amount
of primer when the primer pool is heavily heterogeneous, as each sequence is represented
by only a small fraction of the molecules of the pool, and amounts become limiting in the
20 later amplification cycles. A typical reaction mixture includes: 2jxl of DNA, 25 pmol of
oligonucleotide primer, 2.5 fxl of 10X PCR buffer 1 (Perkin-Elmer, Foster City, CA), 0.4
jlxI of 1.25 fiM dNTP, 0.15 \xX (or 2.5 units) of Taq DNA polymerase (Perkin Elmer,
Foster City, CA) and deionized water to a total volume of 25 jil. Mineral oil is overlaid
and the PCR is performed using a programmable thermal cycler. The length and
25 temperature of each step of a PCR cycle, as well as the number of cycles, is adjusted in
accordance to the stringency requirements in effect. Annealing temperature and timing
are determined both by the efficiency with which a primer is expected to anneal to a
template and the degree of mismatch that is to be tolerated; obviously, when nucleic acid
molecules are simultaneously amplified and mutagenised, mismatch is required, at least
30 in the first round of synthesis. The ability to optimise the stringency of primer annealing
conditions is well within the knowledge of one of moderate skill in the art. An annealing
temperature of between 30 °C and 72 °C is used. Initial denaturation of the template
molecules normally occurs at between 92°C and 99°C for 4 minutes, followed by 20-40
WO 2004/003019 PCT/GB2003/002804
52
cycles consisting of denaturation (94-99°C for 15 seconds to 1 minute), annealing
(temperature determined as discussed above; 1-2 minutes), and extension (72°C for 1-5
minutes, depending on the length of the amplified product). Final extension is generally
for 4 minutes at 72°C, and may be followed by an indefinite (0-24 hour) step at 4°C.
5
C. Combining single variable domains
Domains useful in the invention, once selected, may be combined by a variety of methods
known in the art, including covalent and non-covalent methods.
10
Preferred methods include the use of polypeptide linkers, as described, for example, in
connection with scFv molecules (Bird et al, (1988) Science 242:423-426). Discussion of
suitable linkers is provided in Bird et al Science 242, 423-426; Hudson et al , Journal
Immunol Methods 231 (1999) 177-189; Hudson et al, Proc Nat Acad Sci USA 85, 5879-
15 5883. Linkers are preferably flexible, allowing the two single domains to interact. One
linker example is a (Gly 4 Ser) n linker, where n=l to 8, eg, 2, 3, 4, 5 or 7. The linkers used
in diabodies, which are less flexible, may also be employed (Holliger et al, (1993) PNAS
(USA) 90:6444-6448).
20 In one embodiment, the linker employed is not an immunoglobulin hinge region.
Variable domains may be combined using methods other than linkers. For example, the
use of disulphide bridges, provided through naturally-occurring or engineered cysteine
residues, may be exploited to stabilise V h -V h >Vl-Vl or V H -V L dimers (Reiter et al,
25 (1994) Protein Eng. 7:697-704) or by remodelling the interface between the variable
domains to improve the "fit" and thus the stability of interaction (Ridgeway et al, (1996)
Protein Eng. 7:617-621; Zhu et al, (1997) Protein Science 6:781-788).
Other techniques for joining or stabilising variable domains of immunoglobulins, and in
30 particular antibody V H domains, may be employed as appropriate.
In accordance with the present invention, dual specific ligands can be in "closed"
conformations in solution. A "closed" configuration is that in which the two domains (for
WO 2004/003019 PCT/GB2003/002804
53
example V H and V L ) are present in associated form, such as that of an associated V H -V L
pair which forms an antibody binding site. For example, scFv may be in a closed
conformation, depending on the arrangement of the linker used to link the V H and V L
domains. If this is sufficiently flexible to allow the domains to associate, or rigidly holds
them in the associated position, it is likely that the domains will adopt a closed
conformation.
Similarly, Vh domain pairs and V L domain pairs may exist in a closed conformation.
Generally, this will be a function of close association of the domains, such as by a rigid
linker, in the ligand molecule. Ligands in a closed conformation will be unable to bind
both the molecule which increases the half-life of the ligand and a second target molecule.
Thus, the ligand will typically only bind the second target molecule on dissociation from
the molecule which increases the half-life of the ligand.
Moreover, the construction of V H /V H , Vt/V L or V H /V L dimers without linkers provides
for competition between the domains.
Ligands according to the invention may moreover be in an open conformation. In such a
conformation, the ligands will be able to simultaneously bind both the molecule which
increases the half-life of the ligand and the second target molecule. Typically, variable
domains in an open configuration are (in the case of V H -V L pairs) held far enough apart
for the domains not to interact and form an antibody binding site and not to compete for
binding to their respective epitopes. In the case of V h /Vh or ViTVl dimers, the domains
are not forced together by rigid linkers. Naturally, such domain pairings will not compete
for antigen binding or form an antibody binding site.
Fab fragments and whole antibodies will exist primarily in the closed conformation,
although it will be appreciated that open and closed dual specific ligands are likely to
exist in a variety of equilibria under different circumstances. Binding of the ligand to a
target is likely to shift the balance of the equilibrium towards the open configuration.
Thus, certain ligands according to the invention can exist in two conformations in
solution, one of which (the open form) can bind two antigens or epitopes independently,
WO 2004/003019 PCT/GB2003/002804
54
whilst the alternative conformation (the closed form) can only bind one antigen or
epitope; antigens or epitopes thus compete for binding to the ligand in this conformation.
Although the open form of the dual specific ligand may thus exist in equilibrium with the
5 closed form in solution, it is envisaged that the equilibrium will favour the closed form;
moreover, the open form can be sequestered by target binding into a closed conformation.
Preferably, therefore, certain dual specific ligands of the invention are present in an
equilibrium between two (open and closed) conformations.
10 Dual specific ligands according to the invention may be modified in order to favour an
open or closed conformation. For example, stabilisation of V h ~Vl interactions with
disulphide bonds stabilises the closed conformation. Moreover, linkers used to join the
domains, including Vh domain and Vl domain pairs, may be constructed such that the
open from is favoured; for example, the linkers may sterically hinder the association of
15 the domains, such as by incorporation of large amino acid residues in opportune
locations, or the designing of a suitable rigid structure which will keep the domains
physically spaced apart.
D. Characterisation of the dual-specific ligand .
20
The binding of the dual-specific ligand to its specific antigens or epitopes can be tested by
methods which will be familiar to those skilled in the art and include ELISA. In a
preferred embodiment of the invention binding is tested using monoclonal phage ELISA.
25 Phage ELISA may be performed according to any suitable procedure: an exemplary
protocol is set forth below.
Populations of phage produced at each round of selection can be screened for binding by
ELISA to the selected antigen or epitope, to identify "polyclonal" phage antibodies.
30 Phage from single infected bacterial colonies from these populations can then be screened
by ELISA to identify "monoclonal" phage antibodies. It is also desirable to screen soluble
antibody fragments for binding to antigen or epitope, and this can also be undertaken by
WO 2004/003019 PCT/GB2003/002804
55
ELISA using reagents, for example, against a C- or N-terminal tag (see for example
Winter et al (1994) Aim. Rev. Immunology 12, 433-55 and references cited therein.
The diversity of the selected phage monoclonal antibodies may also be assessed by gel
electrophoresis of PCR products (Marks et al 1991, supra; Nissim et al. 1994 supra),
probing (Tomlinson et al, 1992) J. Mol. Biol. 227, 776) or by sequencing of the vector
DNA.
E. Structure of 'Dual-specific ligands' .
As described above, an antibody is herein defined as an antibody (for example IgG, IgM,
IgA, IgA, IgE) or fragment (Fab, Fv, disulphide linked Fv, scFv, diabody) which
comprises at least one heavy and a light chain variable domain, at least two heavy chain
variable domains or at least two light chain variable domains. It may be at least partly
derived from any species naturally producing an antibody, or created by recombinant
DNA technology; whether isolated from serum, B~cells, hybridomas, transfectomas, yeast
or bacteria).
In a preferred embodiment of the invention the dual-specific ligand comprises at least one
single heavy chain variable domain of an antibody and one single light chain variable
domain of an antibody, or two single heavy or light chain variable domains. For example,
the ligand may comprise a V H /V L pair, a pair of V H domains or a pair of V L domains.
The first and the second variable domains of such a ligand may be on the same
polypeptide chain. Alternatively they may be on separate polypeptide chains. In the case
that they are on the same polypeptide chain they may be linked by a linker, which is
preferentially a peptide sequence, as described above.
The first and second variable domains may be covalently or non-covalently associated. In
the case that they are covalently associated, the covalent bonds may be disulphide bonds.
In the case that the variable domains are selected from V-gene repertoires selected for
instance using phage display technology as herein described, then these variable domains
WO 2004/003019 PCT/GB2003/002804
56
comprise a universal framework region, such that is they may be recognised by a specific
generic ligand as herein defined. The use of universal frameworks, generic ligands and
the like is described in WO99/20749.
Where V-gene repertoires are used variation in polypeptide sequence is preferably located
within the structural loops of the variable domains. The polypeptide sequences of either
variable domain may be altered by DNA shuffling or by mutation in order to enhance the
interaction of each variable domain with its complementary pair. DNA shuffling is
known in the art and taught, for example, by Stemmer, 1994, Nature 370: 389-391 and
U.S. Patent No. 6,297,053, both of which are incorporated herein by reference. Other
methods of mutagenesis are well known to those of skill in the art.
In a preferred embodiment of the invention the 'dual-specific ligand' is a single chain Fv
fragment. In an alternative embodiment of the invention, the 'dual-specific ligand 5
consists of a Fab format.
In a further aspect, the present invention provides nucleic acid encoding at least a 'dual-
specific ligand' as herein defined.
One skilled in the art will appreciate that, depending on the aspect of the invention, both
antigens or epitopes may bind simultaneously to the same antibody molecule.
Alternatively, they may compete for binding to the same antibody molecule. For
example, where both epitopes are bound simultaneously, both variable domains of a dual
specific ligand are able to independently bind their target epitopes. Where the domains
compete, the one variable domain is capable of binding its target, but not at the same time
as the other variable domain binds its cognate target; or the first variable domain is
capable of binding its target, but not at the same time as the second variable domain binds
its cognate target.
The variable regions may be derived from antibodies directed against target antigens or
epitopes. Alternatively they may be derived from a repertoire of single antibody domains
such as those expressed on the surface of filamentous bacteriophage. Selection may be
performed as described below.
WO 2004/003019
57
PCT/GB2003/002804
In general, the nucleic acid molecules and vector constructs required for the performance
of the present invention may be constructed and manipulated as set forth in standard
laboratory manuals, such as Sambrook et aL (1989) Molecular Cloning: A Laboratory
5 Manual, Cold Spring Harbor, USA.
The manipulation of nucleic acids useful in the present invention is typically carried out
in recombinant vectors.
10 Thus in a further aspect, the present invention provides a vector comprising nucleic acid
encoding at least a ' dual-specific ligand 5 as herein defined.
As used herein, vector refers to a discrete element that is used to introduce heterologous
DNA into cells for the expression and/or replication thereof. Methods by which to select
15 or construct and, subsequently, use such vectors are well known to one of ordinary skill in
the art. Numerous vectors are publicly available, including bacterial plasmids,
bacteriophage, artificial chromosomes and episomal vectors. Such vectors may be used
for simple cloning and mutagenesis; alternatively gene expression vector is employed. A
vector of use according to the invention may be selected to accommodate a polypeptide
20 coding sequence of a desired size, typically from 0.25 kilobase (kb) to 40 kb or more in
length A suitable host cell is transformed with the vector after in vitro cloning
manipulations. Each vector contains various functional components, which generally
include a cloning (or "polylinker") site, an origin of replication and at least one selectable
marker gene. If given vector is an expression vector, it additionally possesses one or more
25 of the following: enhancer element, promoter, transcription termination and signal
sequences, each positioned in the vicinity of the cloning site, such that they are
operatively linked to the gene encoding a ligand according to the invention.
Both cloning and expression vectors generally contain nucleic acid sequences that enable
30 the vector to replicate in one or more selected host cells. Typically in cloning vectors, this
sequence is one that enables the vector to replicate independently of the host
chromosomal DNA and includes origins of replication or autonomously replicating
sequences. Such sequences are well known for a variety of bacteria, yeast and viruses.
WO 2004/003019 PCT/GB2003/002804
58
The origin of replication from the plasmid pBR322 is suitable for most Gram-negative
bacteria, the 2 micron plasmid origin is suitable for yeast, and various viral origins (e.g.
SV 40, adenovirus) are useful for cloning vectors in mammalian cells. Generally, the
origin of replication is not needed for mammalian expression vectors unless these are
5 used in mammalian cells able to replicate high levels of DNA, such as COS cells.
Advantageously, a cloning or expression vector may contain a selection gene also
referred to as selectable marker. This gene encodes a protein necessary for the survival or
growth of transformed host cells grown in a selective culture medium. Host cells not
10 transformed with the vector containing the selection gene will therefore not survive in the
culture medium. Typical selection genes encode proteins that confer resistance to
antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline,
complement auxotrophic deficiencies, or supply critical nutrients not available in the
growth media.
15
Since the replication of vectors encoding a ligand according to the present invention is
most conveniently performed in E. coli, an E, co/z-selectable marker, for example, the p~
lactamase gene that confers resistance to the antibiotic ampicillin, is of use. These can be
obtained from E. coli plasmids, such as pBR322 or a pUC plasmid such as pUC18 or
20 pUC19.
Expression vectors usually contain a promoter that is recognised by the host organism and
is operably linked to the coding sequence of interest. Such a promoter may be inducible
or constitutive. The term "operably linked" refers to a juxtaposition wherein the
25 components described are in a relationship permitting them to function in their intended
manner. A control sequence "operably linked" to a coding sequence is ligated in such a
way that expression of the coding sequence is achieved under conditions compatible with
the control sequences.
30 Promoters suitable for use with prokaryotic hosts include, for example, the p-lactamase
and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system
and hybrid promoters such as the tac promoter. Promoters for use in bacterial systems
WO 2004/003019 PCT/GB2003/002804
59
will also generally contain a Shine-Delgarno sequence operably linked to the coding
sequence.
The preferred vectors are expression vectors that enables the expression of a nucleotide
sequence corresponding to a polypeptide library member. Thus, selection with the first
and/or second antigen or epitope can be performed by separate propagation and
expression of a single clone expressing the polypeptide library member or by use of any
selection display system. As described above, the preferred selection display system is
bacteriophage display. Thus, phage or phagemid vectors may be used, eg pITl or pIT2.
Leader sequences useful in the invention include pelB, stll, ompA, phoA, bla and pelA.
One example are phagemid vectors which have an E. colt origin of replication (for
double stranded replication) and also a phage origin of replication (for production of
single-stranded DNA). The manipulation and expression of such vectors is well known in
the art (Hoogenboom and Winter (1992) supra; Nissim et ah (1994) supra). Briefly, the
vector contains a (3-lactamase gene to confer selectivity on the phagemid and a lac
promoter upstream of a expression cassette that consists (N to C terminal) of a pelB
leader sequence (which directs the expressed polypeptide to the periplasmic space), a
multiple cloning site (for cloning the nucleotide version of the library member),
optionally, one or more peptide tag (for detection), optionally, one or more TAG stop
codon and the phage protein pill. Thus, using various suppressor and non-suppressor
strains of E. coli and with the addition of glucose, iso-propyl thio-p-D-galactoside (EPTG)
or a helper phage, such as VCS Ml 3, the vector is able to replicate as a plasmid with no
expression, produce large quantities of the polypeptide library member only or produce
phage, some of which contain at least one copy of the polyp eptide-pIII fusion on their
surface.
Construction of vectors encoding ligands according to the invention employs
conventional ligation techniques. Isolated vectors or DNA fragments are cleaved,
tailored, and religated in the form desired to generate the required vector. If desired,
analysis to confirm that the correct sequences are present in the constructed vector can be
performed in a known fashion. Suitable methods for constructing expression vectors,
preparing in vitro transcripts, introducing DNA into host cells, and performing analyses
for assessing expression and function are known to those skilled in the art. The presence
WO 2004/003019 PCT/GB2003/002804
60
of a gene sequence in a sample is detected, or its amplification and/or expression
quantified by conventional methods, such as Southern or Northern analysis, Western
blotting, dot blotting of DNA, RNA or protein, in situ hybridisation,
immunocytochemistry or sequence analysis of nucleic acid or protein molecules. Those
5 skilled in the art will readily envisage how these methods may be modified, if desired.
Structure of closed conformation multisyecific ligands
According to one aspect of the second configuration of the invention present invention,
10 the two or more non-complementary epitope binding domains are linked so that they are
in a closed conformation as herein defined. Advantageously, they may be further
attached to a skeleton which may, as a alternative, or on addition to a linker described
herein, facilitate the formation and/or maintenance of the closed conformation of the
epitope binding sites with respect to one another.
15
(I) Skeletons
Skeletons may be based on immunoglobulin molecules or may be non-immunoglobulin in
origin as set forth above. Preferred immunoglobulin skeletons as herein defined includes
any one or more of those selected from the following: an immunoglobulin molecule
20 comprising at least (i) the CL (kappa or lambda subclass) domain of an antibody; or (ii) the
CHI domain of an antibody heavy chain; an immunoglobulin molecule comprising the
CHI and CH2 domains of an antibody heavy chain; an immunoglobulin molecule
comprising the CHI, CH2 and CH3 domains of an antibody heavy chain; or any of the
subset (ii) in conjunction with the CL (kappa or lambda subclass) domain of an antibody.
25 A hinge region domain may also be included.. Such combinations of domains may, for
example, mimic natural antibodies, such as IgG or IgM, or fragments thereof, such as Fv,
scFv, Fab or F(ab')2 molecules. Those skilled in the art will be aware that this list is not
intended to be exhaustive.
30 (II) Protein scaffolds
Each epitope binding domain comprises a protein scaffold and one or more CDRs which
are involved in the specific interaction of the domain with one or more epitopes.
Advantageously, an epitope binding domain according to the present invention comprises
WO 2004/003019 PCT/GB2003/002804
61
three CDRs. Suitable protein scaffolds include any of those selected from the group
consisting of the following: those based on immunoglobulin domains, those based on
fibronectin, those based on affibodies, those based on CTLA4, those based on chaperones
such as GroEL, those based on lipocallin and those based on the bacterial Fc receptors
5 SpA and SpD. Those skilled in the art will appreciate that this list is not intended to be
exhaustive.
F: Scaffolds for use in Constructing Dual Specific Ligands
10 i. Selection of the main-chain conformation
The members of the immunoglobulin superfamily all share a similar fold for their
polypeptide chain. For example, although antibodies are highly diverse in terms of their
primary sequence, comparison of sequences and crystallo graphic structures has revealed
that, contrary to expectation, five of the six antigen binding loops of antibodies (HI, H2,
15 LI, L2, L3) adopt a limited number of main-chain conformations, or canonical structures
(Chothia and Lesk (1987) J. Mol Biol, 196: 901; Chothia et al (1989) Nature, 342: 877).
Analysis of loop lengths and key residues has therefore enabled prediction of the main-
chain conformations of HI, H2, LI, L2 and L3 found in the majority of human antibodies
(Chothia et al (1992) J. Mol Biol, 227: 799; Tomlinson et al (1995) EMBO J., 14:
20 4628; Williams et al (1996) J. Mol Biol, 264: 220). Although the H3 region is much
more diverse in terms of sequence, length and structure (due to the use of D segments), it
also forms a limited number of main-chain conformations for short loop lengths which
depend on the length and the presence of particular residues, or types of residue, at key
positions in the loop and the antibody framework (Martin et al (1996) J. Mol Biol, 263:
25 800; Shirai et al (1996) FEBS Letters, 399: 1).
The dual specific ligands of the present invention are advantageously assembled from
libraries of domains, such as libraries of Vh domains and/or libraries of V L domains.
Moreover, the dual specific ligands of the invention may themselves be provided in the
30 form of libraries. In one aspect of the present invention, libraries of dual specific ligands
and/or domains are designed in which certain loop lengths and key residues have been
chosen to ensure that the main-chain conformation of the members is known.
Advantageously, these are real conformations of immunoglobulin superfamily molecules
WO 2004/003019 PCT/GB2003/002804
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found in nature, to minimise the chances that they are non-functional, as discussed
above. Germline V gene segments serve as one suitable basic framework for constructing
antibody or T-cell receptor libraries; other sequences are also of use. Variations may
occur at a low frequency, such that a small number of functional members may possess an
altered main-chain conformation, which does not affect its function.
Canonical structure theory is also of use to assess the number of different main-chain
conformations encoded by ligands, to predict the main-chain conformation based on
ligand sequences and to chose residues for diversification which do not affect the
canonical structure. It is known that, in the human V K domain, the LI loop can adopt one
of four canonical structures, the L2 loop has a single canonical structure and that 90% of
human V K domains adopt one of four or five canonical structures for the L3 loop
(Tomlinson et al (1995) supra); thus, in the V K domain alone, different canonical
structures can combine to create a range of different main-chain conformations. Given
that the V x domain encodes a different range of canonical structures for the LI, L2 and L3
loops and that V K and V* domains can pair with any V H domain which can encode several
canonical structures for the HI and H2 loops, the number of canonical structure
combinations observed for these five loops is very large. This implies that the generation
of diversity in the main-chain conformation may be essential for the production of a wide
range of binding specificities. However, by constructing an antibody library based on a
single known main-chain conformation it has been found, contrary to expectation, that
diversity in the main-chain conformation is not required to generate sufficient diversity to
target substantially all antigens. Even more surprisingly, the single main-chain
conformation need not be a consensus structure - a single naturally occurring
conformation can be used as the basis for an entire library. Thus, in a preferred aspect, the
dual-specific ligands of the invention possess a single known main-chain conformation.
The single main-chain conformation that is chosen is preferably commonplace among
molecules of the immunoglobulin superfamily type in question. A conformation is
commonplace when a significant number of naturally occurring molecules are observed
to adopt it. Accordingly, in a preferred aspect of the invention, the natural occurrence of
the different main-chain conformations for each binding loop of an immunoglobulin
domain are considered separately and then a naturally occurring variable domain is
WO 2004/003019 PCT/GB2003/002804
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chosen which possesses the desired combination of main-chain conformations for the
different loops. If none is available, the nearest equivalent may be chosen. It is preferable
that the desired combination of main-chain conformations for the different loops is
created by selecting germline gene segments which encode the desired main-chain
5 conformations. It is more preferable, that the selected germline gene segments are
frequently expressed in nature, and most preferable that they are the most frequently
expressed of all natural germline gene segments.
In designing dual specific ligands or libraries thereof the incidence of the different main-
10 chain conformations for each of the six antigen binding loops may be considered
separately. For HI, H2, LI, L2 and L3, a given conformation that is adopted by between
20% and 100% of the antigen binding loops of naturally occurring molecules is chosen.
Typically, its observed incidence is above 35% (i.e. between 35% and 100%) and, ideally,
above 50% or even above 65%. Since the vast majority of H3 loops do not have canonical
15 structures, it is preferable to select a main-chain conformation which is commonplace
among those loops which do display canonical structures. For each of the loops, the
conformation which is observed most often in the natural repertoire is therefore selected.
In human antibodies, the most popular canonical structures (CS) for each loop are as
follows: HI - CS 1 (79% of the expressed repertoire), H2 - CS 3 (46%), LI - CS 2 of
20 V K (39%), L2 - CS 1 (100%), L3 - CS 1 of V K (36%) (calculation assumes a k:A, ratio of
70:30, Hood et al (1967) Cold Spring Harbor Symp. Quant. BioL, 48: 133). For H3 loops
that have canonical structures, a CDR3 length (Kabat et al. (1991) Sequences of proteins
of immunological interest, U.S. Department of Health and Human Services) of seven
residues with a salt-bridge from residue 94 to residue 101 appears to be the most
25 common. There are at least 16 human antibody sequences in the EMBL data library with
the required H3 length and key residues to form this conformation and at least two
crystallographic structures in the protein data bank which can be used as a basis for
antibody modelling (2cgr and ltet). The most frequently expressed germline gene
segments that this combination of canonical structures are the Vh segment 3-23 (DP-47),
30 the J H segment JH4b, the V K segment 02/012 (DPK9) and the J K segment J K 1. V H
segments DP45 and DP38 are also suitable. These segments can therefore be used in
combination as a basis to construct a library with the desired single main-chain
conformation.
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PCT/GB2003/002804
Alternatively, instead of choosing the single main-chain conformation based on the
natural occurrence of the different main-chain conformations for each of the binding
loops in isolation, the natural occurrence of combinations of main-chain conformations is
5 used as the basis for choosing the single main-chain conformation. In the case of
antibodies, for example, the natural occurrence of canonical structure combinations for
any two, three, four, five or for all six of the antigen binding loops can be determined.
Here, it is preferable that the chosen conformation is commonplace in naturally occurring
antibodies and most preferable that it observed most frequently in the natural repertoire.
10 Thus, in human antibodies, for example, when natural combinations of the five antigen
binding loops, HI, H2, LI, L2 and L3, are considered, the most frequent combination of
canonical structures is determined and then combined with the most popular
conformation for the H3 loop, as a basis for choosing the single main-chain conformation.
15 ii. Diversification of the canonical sequence
Having selected several known main-chain conformations or, preferably a single
known main-chain conformation, dual specific ligands according to the invention or
libraries for use in the invention can be constructed by varying the binding site of the
molecule in order to generate a repertoire with structural and/or functional diversity. This
20 means that variants are generated such that they possess sufficient diversity in their
structure and/or in their function so that they are capable of providing a range of
activities.
The desired diversity is typically generated by varying the selected molecule at one or
25 more positions. The positions to be changed can be chosen at random or are preferably
selected. The variation can then be achieved either by randomisation, during which the
resident amino acid is replaced by any amino acid or analogue thereof, natural or
synthetic, producing a very large number of variants or by replacing the resident amino
acid with one or more of a defined subset of amino acids, producing a more limited
30 number of variants.
Various methods have been reported for introducing such diversity. Error-prone PGR
(Hawkins et al (1992) J. Mol Biol., 226: 889), chemical mutagenesis (Deng et al (1994)
WO 2004/003019 PCT/GB2003/002804
65
J. Biol Chem., 269: 9533) or bacterial mutator strains (Low et al (1996) J. Mol Biol,
260: 359) can be used to introduce random mutations into the genes that encode the
molecule. Methods for mutating selected positions are also well known in the art and
include the use of mismatched oligonucleotides or degenerate oligonucleotides, with or
5 without the use of PGR. For example, several synthetic antibody libraries have been
created by targeting mutations to the antigen binding loops. The H3 region of a human
tetanus toxoid-binding Fab has been randomised to create a range of new binding
specificities (Barbas et al. (1992) Proc. Natl Acad. Set USA, 89: 4457). Random or
semi-random H3 and L3 regions have been appended to germline V gene segments to
10 produce large libraries with unmutated framework regions (Hoogenboom & Winter
(1992) J. Mol Biol, 227: 381; Barbas et al. (1992) Proc. Natl Acad. Sci. USA, 89: 4457;
Nissim et al (1994) EMBO J., 13: 692; Griffiths et al. (1994) EMBO J., 13: 3245; De
Kruif et al (1995) J. Mol Biol, 248: 97). Such diversification has been extended to
include some or all of the other antigen binding loops (Crameri et al. (1996) Nature Med.,
15 2: 100; Riechmann et al (1995) Bio/Technology, 13: 475; Morphosys, WO97/08320,
supra).
Since loop randomisation has the potential to create approximately more than 10 15
structures for H3 alone and a similarly large number of variants for the other five loops, it
20 is not feasible using current transformation technology or even by using cell free systems
to produce a library representing all possible combinations. For example, in one of the
largest libraries constructed to date, 6 x 10 10 different antibodies, which is only a fraction
of the potential diversity for a library of this design, were generated (Griffiths et al.
(1994) supra).
25
In a preferred embodiment, only those residues which are directly involved in creating or
modifying the desired function of the molecule are diversified. For many molecules, the
function will be to bind a target and therefore diversity should be concentrated in the
target binding site, while avoiding changing residues which are crucial to the overall
30 packing of the molecule or to maintaining the chosen main-chain conformation.
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Diversification of the canonical sequence as it applies to antibody domains
In the case of antibody dual-specific ligands, the binding site for the target is most
often the antigen binding site. Thus, in a highly preferred aspect, the invention provides
libraries of or for the assembly of antibody dual-specific ligands in which only those
5 residues in the antigen binding site are varied. These residues are extremely diverse in the
human antibody repertoire and are known to make contacts in high-resolution
antibody/antigen complexes. For example, in L2 it is known that positions 50 and 53 are
diverse in naturally occurring antibodies and are observed to make contact with the
antigen. In contrast, the conventional approach would have been to diversify all the
10 residues in the corresponding Complementarity Determining Region (CDR1) as defined
by Kabat et al. (1991, supra), some seven residues compared to the two diversified in the
library for use according to the invention. This represents a significant improvement in
terms of the functional diversity required to create a range of antigen binding specificities.
15 In nature, antibody diversity is the result of two processes: somatic recombination of
germline V, D and J gene segments to create a naive primary repertoire (so called
germline and junctional diversity) and somatic hypermutation of the resulting rearranged
V genes. Analysis of human antibody sequences has shown that diversity in the primary
repertoire is focused at the centre of the antigen binding site whereas somatic
20 hypermutation spreads diversity to regions at the periphery of the antigen binding site that
are highly conserved in the primary repertoire (see Tomlinson et al. (1996) J. Mol. Biol,
256: 813). This complementarity has probably evolved as an efficient strategy for
searching sequence space and, although apparently unique to antibodies, it can easily be
applied to other polypeptide repertoires. The residues which are varied are a subset of
25 those that form the binding site for the target. Different (including overlapping) subsets of
residues in the target binding site are diversified at different stages during selection, if
desired.
In the case of an antibody repertoire, an initial 'naive 5 repertoire is created where some,
30 but not all, of the residues in the antigen binding site are diversified. As used herein in
this context, the term "naive" refers to antibody molecules that have no pre-determined
target. These molecules resemble those which are encoded by the immunoglobulin genes
of an individual who has not undergone immune diversification, as is the case with fetal
WO 2004/003019 PCT/GB2003/002804
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and newborn individuals, whose immune systems have not yet been challenged by a
wide variety of antigenic stimuli. This repertoire is then selected against a range of
antigens or epitopes. If required, further diversity can then be introduced outside the
region diversified in the initial repertoire. This matured repertoire can be selected for
5 modified function, specificity or affinity.
The invention provides two different naive repertoires of binding domains for the
construction of dual specific ligands, or a naive library of dual specific ligands, in which
some or all of the residues in the antigen binding site are varied. The "primary" library
10 mimics the natural primary repertoire, with diversity restricted to residues at the centre of
the antigen binding site that are diverse in the germline V gene segments (germline
diversity) or diversified during the recombination process (junctional diversity). Those
residues which are diversified include, but are not limited to, H50, H52, H52a, H53, H55,
H56, H58, H95, H96, H97, H98, L50, L53, L91, L92, L93, L94 and L96. In the "somatic"
15 library, diversity is restricted to residues that are diversified during the recombination
process (junctional diversity) or are highly somatically mutated). Those residues which
are diversified include, but are not limited to: H31, H33, H35, H95, H96, H97, H98, L30,
L31, L32, L34 and L96. All the residues listed above as suitable for diversification in
these libraries are known to make contacts in one or more antibody-antigen complexes.
20 Since in both libraries, not all of the residues in the antigen binding site are varied,
additional diversity is incorporated during selection by varying the remaining residues, if
it is desired to do so. It shall be apparent to one skilled in the art that any subset of any of
these residues (or additional residues which comprise the antigen binding site) can be
used for the initial and/or subsequent diversification of the antigen binding site.
25
In the construction of libraries for use in the invention, diversification of chosen positions
is typically achieved at the nucleic acid level, by altering the coding sequence which
specifies the sequence of the polypeptide such that a number of possible amino acids (all
20 or a subset thereof) can be incorporated at that position. Using the IUPAC
30 nomenclature, the most versatile codon is NNK, which encodes all amino acids as well as
the TAG stop codon. The NNK codon is preferably used in order to introduce the
required diversity. Other codons which achieve the same ends are also of use, including
WO 2004/003019 PCT/GB2003/002804
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the NNN codon, which leads to the production of the additional stop codons TGA and
TAA.
A feature of side-chain diversity in the antigen binding site of human antibodies is a
5 pronounced bias which favours certain amino acid residues. If the amino acid
composition of the ten most diverse positions in each of the Vh, V k and Vx regions are
summed, more than 76% of the side-chain diversity comes from only seven different
residues, these being, serine (24%), tyrosine (14%), asparagine (11%), glycine (9%),
alanine (7%), aspartate (6%) and threonine (6%). This bias towards hydrophilic residues
10 and small residues which can provide main-chain flexibility probably reflects the
evolution of surfaces which are predisposed to binding a wide range of antigens or
epitopes and may help to explain the required promiscuity of antibodies in the primary
repertoire.
15 Since it is preferable to mimic this distribution of amino acids, the distribution of amino
acids at the positions to be varied preferably mimics that seen in the antigen binding site
of antibodies. Such bias in the substitution of amino acids that permits selection of certain
polypeptides (not just antibody polypeptides) against a range of target antigens is easily
applied to any polypeptide repertoire. There are various methods for biasing the amino
20 acid distribution at the position to be varied (including the use of tri-nucleotide
mutagenesis, see WO97/08320), of which the preferred method, due to ease of synthesis,
is the use of conventional degenerate codons. By comparing the amino acid profile
encoded by all combinations of degenerate codons (with single, double, triple and
quadruple degeneracy in equal ratios at each position) with the natural amino acid use it is
25 possible to calculate the most representative codon. The codons (AGT)(AGC)T,
(AGT)(AGC)C and (AGT)(AGC)(CT) - that is, DVT, DVC and DVY, respectively using
IUPAC nomenclature - are those closest to the desired amino acid profile: they encode
22% serine and 11% tyrosine, asparagine, glycine, alanine, aspartate, threonine and
cysteine. Preferably, therefore, libraries are constructed using either the DVT, DVC or
30 DVY codon at each of the diversified positions.
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G: Antigens capable of increasing ligand half-life
PCT/GB2003/002804
The dual specific ligands according to the invention, in one configuration thereof, are
capable of binding to one or more molecules which can increase the half-life of the ligand
5 in vivo. Typically, such molecules are polypeptides which occur naturally in vivo and
which resist degradation or removal by endogenous mechanisms which remove unwanted
material from the organism. For example, the molecule which increases the half-life of
the organism may be selected from the following:
10 Proteins from the extracellular matrix; for example collagen, laminins, integrins and
fibronectin. Collagens are the major proteins of the extracellular matrix. About 15 types
of collagen molecules are currently known, found in different parts of the body, eg type I
collagen (accounting for 90% of body collagen) found in bone, skin, tendon, ligaments,
cornea, internal organs or type II collagen found in cartilage, invertebral disc, notochord,
1 5 vitreous humour of the eye.
Proteins found in blood, including:
Plasma proteins such as fibrin, a-2 macroglobulin, serum albumin, fibrinogen A,
20 fibrinogen B, serum amyloid protein A, heptaglobin, profilin, ubiquitin, uteroglobulin and
/3-2-micro globulin;
Enzymes and inhibitors such as plasminogen, lysozyme, cystatin C, alpha- 1 -antitrypsin
and pancreatic trypsin inhibitor. Plasminogen is the inactive precursor of the trypsin-like
25 serine protease plasmin. It is normally found circulating through the blood stream. When
plasminogen becomes activated and is converted to plasmin, it unfolds a potent enzymatic
domain that dissolves the fibrinogen fibers that entgangle the blood cells in a blood clot.
This is called fibrinolysis.
30 Immune system proteins, such as IgE, IgG, IgM.
Transport proteins such as retinol binding protein, ol-1 microglobulin.
WO 2004/003019 PCT/GB2003/002804
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Defensins such as beta-defensin 1 , Neutrophil defensins 1 ,2 and 3 .
Proteins found at the blood brain barrier or in neural tissues, such as melanocortin
receptor, myelin, ascorbate transporter.
5
Transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins (see
US5977307);
brain capillary endothelial cell receptor, transferrin, transferrin receptor, insulin, insulin-
10 like growth factor 1 (IGF 1) receptor, insulin-like growth factor 2 (IGF 2) receptor,
insulin receptor.
Proteins localised to the kidney, such as polycystin, type IV collagen, organic anion
transporter Kl, Heymann's antigen.
15
Proteins localised to the liver, for example alcohol dehydrogenase, G250.
Blood coagulation factor X
al antitrypsin
20 HNF la
Proteins localised to the lung, such as secretory component (binds IgA).
Proteins localised to the Heart, for example HSP 27. This is associated with dilated
*
25 cardiomyopathy.
Proteins localised to the skin, for example keratin.
Bone specific proteins, such as bone morphogenic proteins (BMPs), which are a subset of
30 the transforming growth factor (3 superfamily that demonstrate osteogenic activity.
Examples include BMP-2, -4, -5, -6, -7 (also referred to as osteogenic protein (OP-1) and
-8 (OP-2).
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Tumour specific proteins, including human trophoblast antigen, herceptin receptor,
oestrogen receptor, cathepsins eg cathepsin B (found in liver and spleen).
Disease-specific proteins, such as antigens expressed only on activated T-cells: including
LAG-3 (lymphocyte activation gene), osteoprotegerin ligand (OPGL) see Nature 402,
304-309; 1999, OX40 (a member of the TNF receptor family, expressed on activated T
cells and the only costimulatory T cell molecule known to be specifically up-regulated in
human T cell leukaemia virus type-I (HTLV-I)-producing cells.) See J Immunol 2000
Jul l;165(l):263-70\ Metalloproteases (associated with arthritis/cancers), including
CG6512 Drosophila, human paraplegin, human FtsH, human AFG3L2, murine ftsH;
angiogenic growth factors, including acidic fibroblast growth factor (FGF-1), basic
fibroblast growth factor (FGF-2), Vascular endothelial growth factor / vascular
permeability factor (VEGF/VPF), transforming growth factor-a (TGF a), tumor necrosis
factor-alpha (TNF-o:), angiogenin, interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-
derived endothelial growth factor (PD-ECGF), placental growth factor (P1GF), midkine
platelet-derived growth factor-BB (PDGF), fractalkine.
Stress proteins (heat shock proteins)
HSPs are normally found intracellularly. When they are found extracellularly, it is an
indicator that a cell has died and spilled out its contents. This unprogrammed cell death
(necrosis) only occurs when as a result of trauma, disease or injury and therefore in vivo,
extracellular HSPs trigger a response from the immune system that will fight infection
and disease. A dual specific which binds to extracellular HSP can be localised to a
disease site.
Proteins involved in Fc transport
Brambell receptor (also known as FcRB)
This Fc receptor has two functions, both of which are potentially useful for delivery
The functions are
(1) The transport of IgG from mother to child across the placenta
(2) the protection of IgG from degradation thereby prolonging its serum half life of
IgG. It is thought that the receptor recycles IgG from endosome.
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See Holliger et al 9 Nat Biotechnol 1997 Jul;15(7):632-6
PCT/GB2003/002804
Ligands according to the invention may designed to be specific for the above targets
without requiring any increase in or increasing half life in vivo. For example, ligands
5 according to the invention can be specific for targets selected from the foregoing which
are tissue-specific, thereby enabling tissue-specific targeting of the dual specific ligand,
or a dAb monomer that binds a tissue-specific therapeutically relevant target, irrespective
of any increase in half-life, although this may result. Moreover, where the ligand or dAb
monomer targets kidney or liver, this may redirect the ligand or dAb monomer to an
10 alternative clearance pathway in vivo (for example, the ligand may be directed away from
liver clearance to kidney clearance).
H: Use of multispecific ligands according to the second configuration of the
invention
15
Multispecific ligands according to the method of the second configuration of the present
invention may be employed in in vivo therapeutic and prophylactic applications, in vitro
and in vivo diagnostic applications, in vitro assay and reagent applications, and the like.
For example antibody molecules may be used in antibody based assay techniques, such as
20 ELISA techniques, according to methods known to those skilled in the art.
As alluded to above, the multispecific ligands according to the invention are of use in
diagnostic, prophylactic and therapeutic procedures. Multispecific antibodies according to
the invention are of use diagnostically in Western analysis and in situ protein detection by
25 standard immunohistochemical procedures; for use in these applications, the ligands may
be labelled in accordance with techniques known to the art. In addition, such antibody
polypeptides may be used preparatively in affinity chromatography procedures, when
complexed to a chromatographic support, such as a resin. All such techniques are well
knowm to one of skill in the art.
30
Diagnostic uses of the closed conformation multispecific ligands according to the
invention include homogenous assays for analytes which exploit the ability of closed
conformation multispecific ligands to bind two targets in competition, such that two
WO 2004/003019 PCT/GB2003/002804
73
targets cannot bind simultaneously (a closed conformation), or alternatively their ability
to bind two targets simultaneously (an open conformation).
A true homogenous immunoassay format has been avidly sought by manufacturers of
5 diagnostics and research assay systems used in drug discovery and development. The
main diagnostics markets include human testing in hospitals, doctor's offices and clinics,
commercial reference laboratories, blood banks, and the home, non-human diagnostics
(for example food testing, water testing, environmental testing, bio-defence, and
veterinary testing), and finally research (including drug development; basic research and
1 o academic research) .
At present all these markets utilise immunoassay systems that are built around
chemiluminescent, ELISA, fluorescence or in rare cases radio-immunoassay
technologies. Each of these assay formats requires a separation step (separating bound
15 from un-bound reagents). In some cases, several separation steps are required. Adding
these additional steps adds reagents and automation, takes time, and affects the ultimate
outcome of the assays. In human diagnostics, the separation step may be automated,
which masks the problem, but does not remove it. The robotics, additional reagents,
additional incubation times, and the like add considerable cost and complexity. In drug
20 development, such as high throughput screening, where literally millions of samples are
tested at once, with very low levels of test molecule, adding additional separation steps
can eliminate the ability to perform a screen. However, avoiding the separation creates
too much noise in the read out. Thus, there is a need for a true homogenous format that
provides sensitivities at the range obtainable from present assay formats. Advantageously,
25 an assay possesses fully quantitative read-outs with high sensitivity and a large dynamic
range. Sensitivity is an important requirement, as is reducing the amount of sample
required. Both of these features are features that a homogenous system offers. This is
very important in point of care testing, and in drug development where samples are
precious. Heterogenous systems, as currently available in the art, require large quantities
30 of sample and expensive reagents
Applications for homogenous assays include cancer testing, where the biggest assay is
that for Prostate Specific Antigen, used in screening men for prostate cancer. Other
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74
applications include fertility testing, which provides a series of tests for women
attempting to conceive including beta-hcg for pregnancy. Tests for infectious diseases,
including hepatitis, HIV, rubella, and other viruses and microorganisms and sexually
transmitted diseases. Tests are used by blood banks, especially tests for HIV, hepatitis A,
5 B, C, non A non B. Therapeutic drug monitoring tests include monitoring levels of
prescribed drugs in patients for efficacy and to avoid toxicity, for example digoxin for
arrhythmia, and phenobarbital levels in psychotic cases; theophylline for asthma.
Diagnostic tests are moreover useful in abused drug testing, such as testing for cocaine,
marijuana and the like. Metabolic tests are used for measuring thyroid function, anaemia
10 and other physiological disorders and functions.
The homogenous immunoassay format is moreover useful in the manufacture of standard
clinical chemistry assays. The inclusion of immunoassays and chemistry assays on the
same instrument is highly advantageous in diagnostic testing. Suitable chemical assays
15 include tests for glucose, cholesterol, potassium, and the like.
A further major application for homogenous immunoassays is drug discovery and
development: high throughput screening includes testing combinatorial chemistry
libraries versus targets in ultra high volume. Signal is detected, and positive groups then
20 split into smaller groups, and eventually tested in cells and then animals. Homogenous
assays may be used in all these types of test. In drug development, especially animal
studies and clinical trials heavy use of immunoassays is made. Homogenous assays
greatly accelerate and simplify these procedures. Other Applications include food and
beverage testing: testing meat and other foods for E. coli, salmonella, etc; water testing,
25 including testing at water plants for all types of contaminants including E. coli; and
veterinary testing.
In a broad embodiment, the invention provides a binding assay comprising a detectable
agent which is bound to a closed conformation multispecific ligand according to the
30 invention, and whose detectable properties are altered by the binding of an analyte to said
closed conformation multispecific ligand. Such an assay may be configured in several
different ways, each exploiting the above properties of closed conformation multispecific
ligands.
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PCT/GB2003/002804
The assay relies on the direct or indirect displacement of an agent by the analyte, resulting
in a change in the detectable properties of the agent. For example, where the agent is an
enzyme which is capable of catalysing a reaction which has a detectable end-point, said
5 enzyme can be bound by the ligand such as to obstruct its active site, thereby inactivating
the enzyme. The analyte, which is also bound by the closed conformation multispecific
ligand, displaces the enzyme, rendering it active through freeing of the active site. The
enzyme is then able to react with a substrate, to give rise to a detectable event. In an
alternative embodiment, the ligand may bind the enzyme outside of the active site,
10 influencing the conformation of the enzyme and thus altering its activity. For example,
the structure of the active site may be constrained by the binding of the ligand, or the
binding of cofactors necessary for activity may be prevented.
The physical implementation of the assay may take any form known in the art. For
15 example, the closed conformation multispecific ligand/enzyme complex may be provided
on a test strip; the substrate may be provided in a different region of the test strip, and a
solvent containing the analyte allowed to migrate through the ligand/enzyme complex,
displacing the enzyme, and carrying it to the substrate region to produce a signal.
Alternatively, the ligand/enzyme complex may be provided on a test stick or other solid
20 phase, and dipped into an analyte/substrate solution, releasing enzyme into the solution in
response to the presence of analyte.
Since each molecule of analyte potentially releases one enzyme molecule, the assay is
quantitative, with the strength of the signal generated in a given time being dependent on
25 the concentration of analyte in the solution.
Further configurations using the analyte in a closed conformation are possible. For
example, the closed conformation multispecific ligand may be configured to bind an
enzyme in an allosteric site, thereby activating the enzyme. In such an embodiment, the
30 enzyme is active in the absence of analyte. Addition of the analyte displaces the enzyme
and removes allosteric activation, thus inactivating the enzyme.
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In the context of the above embodiments which employ enzyme activity as a measure of
the analyte concentration, activation or inactivation of the enzyme refers to an increase or
decrease in the activity of the enzyme, measured as the ability of the enzyme to catalyse a
signal-generating reaction. For example, the enzyme may catalyse the conversion of an
undetectable substrate to a detectable form thereof. For example, horseradish peroxidase
is widely used in the art together with chromogenic or chemiluminescent substrates,
which are available commercially. The level of increase or decrease of the activity of the
enzyme may between 10% and 100%, such as 20%, 30%, 40%, 50%, 60%, 70%, 80% or
90%; in the case of an increase in activity, the increase may be more than 100%, i.e.
200%, 300%, 500% or more, or may not be measurable as a percentage if the baseline
activity of the inhibited enzyme is undetectable.
In a further configuration, the closed conformation multispecific ligand may bind the
substrate of an enzyme/substrate pair, rather than the enzyme. The substrate is therefore
unavailable to the enzyme until released from the closed conformation multispecific
ligand through binding of the analyte. The implementations for this configuration are as
for the configurations which bind enzyme.
Moreover, the assay may be configured to bind a fluorescent molecule, such as a
fluorescein or another fluorophore, in a conformation such that the fluorescence is
quenched on binding to the ligand. In this case, binding of the analyte to the ligand will
displace the fluorescent molecule, thus producing a signal. Alternatives to fluorescent
molecules which are useful in the present invention include luminescent agents, such as
luciferin/luciferase, and chromogenic agents, including agents commonly used in
immunoassays such as HRP.
Therapeutic and prophylactic uses of multispecific ligands prepared according to the
invention involve the administration of ligands according to the invention to a recipient
mammal, such as a human. Multi-specificity can allow antibodies to bind to multimeric
antigen with great avidity. Multispecific ligands can allow thecross- linking of two
antigens, for example in recruiting cytotoxic T-cells to mediate the killing of tumour cell
lines.
WO 2004/003019 PCT/GB2003/002804
77
Substantially pure ligands or binding proteins thereof, for example dAb monomers, of at
least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to
99% or more homogeneity is most preferred for pharmaceutical uses, especially when the
mammal is a human. Once purified, partially or to homogeneity as desired, the ligands
5 may be used diagnostically or therapeutically (including extracorporeally) or in
developing and performing assay procedures, immunofluorescent stainings and the like
(Lefkovite and Pemis, (1979 and 1981) Immunological Methods, Volumes I and II,
Academic Press, NY).
10 The ligands or binding proteins thereof, for example dAb monomers, of the present
invention will typically find use in preventing, suppressing or treating inflammatory
states, allergic hypersensitivity, cancer, bacterial or viral infection, and autoimmune
disorders (which include, but are not limited to, Type I diabetes, asthma, multiple
sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and
15 myasthenia gravis).
In the instant application, the term "prevention" involves administration of the protective
composition prior to the induction of the disease. "Suppression" refers to administration
of the composition after an inductive event, but prior to the clinical appearance of the
20 disease. "Treatment" involves administration of the protective composition after disease
symptoms become manifest.
Animal model systems which can be used to screen the effectiveness of the antibodies or
binding proteins thereof in protecting against or treating the disease are available.
25 Methods for the testing of systemic lupus erythematosus (SLE) in susceptible mice are
known in the art (Knight et al (1978) J. Exp. Med., 147: 1653; Reinersten et al (1978)
New Eng. J. Med., 299: 515). Myasthenia Gravis (MG) is tested in SJL/J female mice by
inducing the disease with soluble AchR protein from another species (Lmdstrom et al
(1988) Adv. Immunol, 42: 233). Arthritis is induced in a susceptible strain of mice by
30 injection of Type II collagen (Stuart et al. (1984) Ann. Rev. Immunol, 42: 233). A model
by which adjuvant arthritis is induced in susceptible rats by injection of mycobacterial
heat shock protein has been described (Van Eden et al (1988) Nature, 331: 171).
Thyroiditis is induced in mice by administration of thyroglobulin as described (Maron et
WO 2004/003019 PCT/GB2003/002804
78
al (1980) J. Exp. Med., 152: 1115). Insulin dependent diabetes mellitus (IDDM) occurs
naturally or can be induced in certain strains of mice such as those described by
Kanasawa et al (1984) Diabetologia, 27: 113. EAE in mouse and rat serves as a model
for MS in human. In this model, the demyelinating disease is induced by administration
of myelin basic protein (see Paterson (1986) Textbook of Immunopathology, Mischer et
al 9 eds., Grune and Stratton, New York, pp. 179-213; McFarlin et al (1973) Science,
179: 478: and Satoh et al (1987) J. Immunol, 138: 179).
Generally, the present ligands will be utilised in purified form together with
pharmacologically appropriate carriers. Typically, these carriers include aqueous or
alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or
buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable
adjuvants, if necessary to keep a polypeptide complex in suspension, maybe chosen from
thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.
Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers,
such as those based on Ringer's dextrose. Preservatives and other additives, such as
antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack
(1982) Remington's Pharmaceutical Sciences, 16th Edition).
The ligands of the present invention may be used as separately administered compositions
or in conjunction with other agents. These can include various immunotherapeutic drugs,
such as cylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins.
Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents
in conjunction with the ligands of the present invention, or even combinations of lignds
according to the present invention having different specificities, such as ligands selected
using different target antigens or epitopes, whether or not they are pooled prior to
administration.
The route of administration of pharmaceutical compositions according to the invention
may be any of those commonly known to those of ordinary skill in the art. For therapy,
including without limitation immunotherapy, the selected ligands thereof of the invention
WO 2004/003019 PCT/GB2003/002804
79
can be administered to any patient in accordance with standard techniques. The
administration can be by any appropriate mode, including parenterally, intravenously,
intramuscularly, intraperitoneal^, transdermally, via the pulmonary route, or also,
appropriately, by direct infusion with a catheter. The dosage and frequency of
5 administration will depend on the age, sex and condition of the patient, concurrent
administration of other drugs, counterindications and other parameters to be taken into
account by the clinician.
The ligands of this invention can be lyophilised for storage and reconstituted in a suitable
10 carrier prior to use. This technique has been shown to be effective with conventional
immunoglobulins and art-known lyophilisation and reconstitution techniques can be
employed. It will be appreciated by those skilled in the art that lyophilisation and
reconstitution can lead to varying degrees of antibody activity loss (e.g. with conventional
immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies)
15 and that use levels may have to be adjusted upward to compensate.
The compositions containing the present ligands or a cocktail thereof can be administered
for prophylactic and/or therapeutic treatments. In certain therapeutic applications, an
adequate amount to accomplish at least partial inhibition, suppression, modulation,
20 killing, or some other measurable parameter, of a population of selected cells is defined as
a "therapeutically-effective dose". Amounts needed to achieve this dosage will depend
upon the severity of the disease and the general state of the patient's own immune system,
but generally range from 0.005 to 5.0 mg of ligand, e.g. antibody, receptor (e.g. a T-cell
receptor) or binding protein thereof per kilogram of body weight, with doses of 0.05 to
25 2.0 mg/kg/dose being more commonly used. For prophylactic applications, compositions
containing the present ligands or cocktails thereof may also be administered in similar or
slightly lower dosages.
Treatment performed using the compositions described herein is considered "effective" if
30 one or more symptoms is reduced (e.g., by at least 10% or at least one point on a clinical
assessment scale), relative to such symptoms present before treatment, or relative to such
symptoms in an individual (human or model animal) not treated with such composition.
Symptoms will obviously vary depending upon the disease or disorder targeted, but can
WO 2004/003019 PCT/GB2003/002804
80
be measured by an ordinarily skilled clinician or technician. Such symptoms can be
measured, for example, by monitoring the level of one or more biochemical indicators of
the disease or disorder (e.g., levels of an enzyme or metabolite correlated with the
disease, ' affected cell numbers, etc.), by monitoring physical manifestations (e.g.,
5 inflammation, tumor size, etc.), or by an accepted clinical assessment scale, for example,
the Expanded Disability Status Scale (for multiple sclerosis), the Irvine Inflammatory
Bowel Disease Questionnaire (32 point assessment evaluates quality of life with respect
to bowel function, systemic symptoms, social function and emotional status - score ranges
from 32 to 224, with higher scores indicating a better quality of life), the Quality of Life
10 Rheumatoid Arthritis Scale, or other accepted clinical assessment scale as known in the
field. A sustained (e.g., one day or more, preferably longer) reduction in disease or
disorder symptoms by at least 10% or by one or more points on a given clinical scale is
indicative of "effective" treatment. Similarly, prophylaxis performed using a composition
as described herein is "effective" if the onset or severity of one or more symptoms is
15 delayed, reduced or abolished relative to such symptoms in a similar individual (human or
animal model) not treated with the composition.
A composition containing a ligand or cocktail thereof according to the present invention
may be utilised in prophylactic and therapeutic settings to aid in the alteration,
20 inactivation, killing or removal of a select target cell population in a mammal. In addition,
the selected repertoires of polypeptides described herein may be used extracorporeally or
in vitro selectively to kill, deplete or otherwise effectively remove a target cell population
from a heterogeneous collection of cells. Blood from a mammal may be combined
extracorporeally with the ligands, e.g. antibodies, cell-surface receptors or binding
25 proteins thereof whereby the undesired cells are killed or otherwise removed from the
blood for return to the mammal in accordance with standard techniques.
I: Use of half-life enhanced dual-specific ligands according to the invention
30
Dual-specific ligands according to the method of the present invention may be employed
in in vivo therapeutic and prophylactic applications, in vivo diagnostic applications and
the like.
WO 2004/003019
81
PCT/GB2003/002804
Therapeutic and prophylactic uses of dual-specific ligands prepared according to the
invention involve the administration of ligands according to the invention to a recipient
mammal, such as a human. Dual specific antibodies according to the invention comprise
5 at least one specificity for a half-life enhancing molecule; one or more further specificities
may be directed against target molecules. For example, a dual-specific IgG may be
specific for four epitopes, one of which is on a half-life enhancing molecule. Dual-
specificity can allow antibodies to bind to multimeric antigen with great avidity. Dual-
specific antibodies can allow the cross-linking of two antigens, for example in recruiting
10 cytotoxic T-cells to mediate the killing of tumour cell lines.
Substantially pure ligands or binding proteins thereof, such as dAb monomers, of at least
90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or
more homogeneity is most preferred for pharmaceutical uses, especially when the
15 mammal is a human. Once purified, partially or to homogeneity as desired, the ligands
may be used diagnostically or therapeutically (including extracorporeally) or in
developing and performing assay procedures, immunofluorescent stainings and the like
(Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II,
Academic Press, NY).
20
The ligands of the present invention will typically find use in preventing, suppressing or
treating inflammatory states, allergic hypersensitivity, cancer, bacterial or viral infection,
and autoimmune disorders (which include, but are not limited to, Type I diabetes,
multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease
25 and myasthenia gravis).
In the instant application, the term "prevention" involves administration of the protective
composition prior to the induction of the disease. "Suppression" refers to administration
of the composition after an inductive event, but prior to the clinical appearance of the
30 disease. "Treatment" involves administration of the protective composition after disease
symptoms become manifest.
WO 2004/003019 PCT/GB2003/002804
82
Animal model systems which can be used to screen the effectiveness of the dual specific
ligands in protecting against or treating the disease are available. Methods for the testing
of systemic lupus erythematosus (SLE) in susceptible mice are known in the art (Knight
et al (1978) J. Exp. Med., 147: 1653; Reinersten et al (1978) New Eng. J. Med, 299:
5 515). Myasthenia Gravis (MG) is tested in SJL/J female mice by inducing the disease
with soluble AchR protein from another species (Lindstrom et al (1988) Adv. Immunol,
42: 233). Arthritis is induced in a susceptible strain of mice by injection of Type II
collagen (Stuart et al (1984) Ann. Rev. Immunol, 42: 233). A model by which adjuvant
arthritis is induced in susceptible rats by injection of mycobacterial heat shock protein has
10 been described (Van Eden et al. (1988) Nature, 331: 171). Thyroiditis is induced in mice
by administration of thyroglobulin as described (Maron et al (1980) J. Exp. Med., 152:
1115). Insulin dependent diabetes mellitus (IDDM) occurs naturally or can be induced in
certain strains of mice such as those described by Kanasawa et al (1984) Diabetologia,
27: 113. EAE in mouse and rat serves as a model for MS in human. In this model, the
15 demyelinating disease is induced by administration of myelin basic protein (see Paterson
(1986) Textbook of Immunopathology, Mischer et al, eds., Grune and Stratton, New
York, pp. 179-213; McFarlin et al (1973) Science, 179: 478: and Satoh et al (1987) J.
Immunol, 138: 179).
20 Dual specific ligands according to the invention and dAb monomers able to bind to
extracellular targets involved in endocytosis (e.g. Clathrin) enable dual specific ligands to
be endocytosed, enabling another specificity able to bind to an intracellular target to be
delivered to an intracellular environment. This strategy requires a dual specific ligand
with physical properties that enable it to remain functional inside the cell. Alternatively,
25 if the final destination intracellular compartment is oxidising, a well folding ligand may
not need to be disulphide free.
Generally, the present dual specific ligands will be utilised in purified form together with
pharmacologically appropriate carriers. Typically, these carriers include aqueous or
30 alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or
buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable
WO 2004/003019 PCT/GB2003/002804
83
adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen
from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and
alginates.
5 Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishes,
such as those based on Ringer's dextrose. Preservatives and other additives, such as
antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack
(1982) Remington's Pharmaceutical Sciences,, 16th Edition).
10 The ligands of the present invention may be used as separately administered compositions
or in conjunction with other agents. These can include various immunotherapeutic drugs,
such as cylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins.
Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents
in conjunction with the ligands of the present invention.
15
The route of administration of pharmaceutical compositions according to the invention
may be any of those commonly known to those of ordinary skill in the art. For therapy,
including without limitation immunotherapy, the ligands of the invention can be
administered to any patient in accordance with standard techniques. The administration
20 can be by any appropriate mode, including parenterally, intravenously, intramuscularly,
intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct
infusion with a catheter. The dosage and frequency of administration will depend on the
age, sex and condition of the patient, concurrent administration of other drugs,
counterindications and other parameters to be taken into account by the clinician.
25
The ligands of the invention can be lyophilised for storage and reconstituted in a suitable
carrier prior to use. This technique has been shown to be effective with conventional
immunoglobulins and art-known lyophilisation and reconstitution techniques can be
employed. It will be appreciated by those skilled in the art that lyophilisation and
30 reconstitution can lead to varying degrees of antibody activity loss (e.g. with conventional
immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies)
and that use levels may have to be adjusted upward to compensate.
WO 2004/003019 PCT/GB2003/002804
84
The compositions containing the present ligands or a cocktail thereof can be
administered for prophylactic and/or therapeutic treatments. In certain therapeutic
applications, an adequate amount to accomplish at least partial inhibition, suppression,
modulation, killing, or some other measurable parameter, of a population of selected cells
5 is defined as a "therapeutically-effective dose". Amounts needed to achieve this dosage
will depend upon the severity of the disease and the general state of the patient's own
immune system, but generally range from 0.005 to 5.0 mg of ligand per kilogram of body
weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. For
prophylactic applications, compositions containing the present ligands or cocktails thereof
10 may also be administered in similar or slightly lower dosages.
A composition containing a ligand according to the present invention may be utilised in
prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or
removal of a select target cell population in a mammal
15
In addition, the selected repertoires of polypeptides described herein may be used
extracorporeal^ or in vitro selectively to kill, deplete or otherwise effectively remove a
target cell population from a heterogeneous collection of cells. Blood from a mammal
may be combined extracorporeal!/ with the ligands, e.g. antibodies, cell- surface
20 receptors or binding proteins thereof whereby the undesired cells are killed or otherwise
removed from the blood for return to the mammal in accordance with standard
techniques.
The invention is further described, for the purposes of illustration only, in the following
25 examples. As used herein, for the purposes of dAb nomenclature, human TNFoc is
referred to as TAR1 and human TNFoc receptor 1 (p55 receptor) is referred to as TAR2.
Example 1. Selection of a dual specific scFv antibody (K8) directed against human
30 serum albumin (HSA) and (3-galactosidase (p -gal)
This example explains a method for making a dual specific antibody directed against p-
gal and HSA in which a repertoire of V K variable domains linked to a germline (dummy)
10
WO 2004/003019 PCT/GB2003/002804
85
V H domain is selected for binding to (3-gal and a repertoire of V H variable domains
linked to a germline (dummy) V K domain is selected for binding to HSA. The selected
variable Vh HSA and V K (3-gal domains are then combined and the antibodies selected for
binding to P-gal and HSA. HSA is a half-life increasing protein found in human blood.
Four human phage antibody libraries were used in this experiment.
Library 1 Germline V K /DVT V H 8.46 x 1 0?
Library 2 Germline V K /NNK V H 9.64 x 10 7
Library 3 Germline V H /D VT V K 1 .47 x 1 0 8
Library 4 Germline V H /NNK V K 1 .45 x 1 0 s
All libraries are based on a single human framework for Vh (V3-23/DP47 and JH4b) and
V K (012/02/DPK9 and J K 1) with side chain diversity incorporated in complementarity
15 determining regions (CDR2 and CDR3).
Library 1 and Library 2 contain a dummy V K sequence, whereas the sequence of Vh is
diversified at positions H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97 and H98
(DVT or NNK encoded, respectively) (Figure 1). Library 3 and Library 4 contain a
20 dummy Vh sequence, whereas the sequence of V K is diversified at positions L50, L53,
L91, L92, L93, L94 and L96 (DVT or NNK encoded, respectively) (Figure 1). The
libraries are in phagemid pIT2/ScFv format (Figure 2) and have been preselected for
binding to generic ligands, Protein A and Protein L, so that the majority of clones in the
unselected libraries are functional. The sizes of the libraries shown above correspond to
25 the sizes after preselection. Library 1 and Library 2 were mixed prior to selections on
antigen to yield a single Vn/dummy V K library and Library 3 and Library 4 were mixed
to form a single V K /dummy Vh library.
Three rounds of selections were performed on (3-gal using V K /dummy V H library and
30 three rounds of selections were performed on HSA using V H /dummy V K library. In the
WO 2004/003019 PCT/GB2003/002804
86
case of P-gal the phage titres went up from 1.1 x 10 6 in the first round to 2.0 x 10 8 in the
third round. In the case of HSA the phage titres went up from 2 x 10 4 in the first round to
1.4 x 10 9 in the third round. The selections were performed as described by Griffith et aL,
(1993), except that KM13 helper phage (which contains a pill protein with a protease
5 cleavage site between the D2 and D3 domains) was used and phage were eluted with 1
mg/ml trypsin in PBS. The addition of trypsin cleaves the pill proteins derived from the
helper phage (but not those from the phagemid) and elutes bound scFv-phage fusions by
cleavage in the c-myc tag (Figure 2), thereby providing a further enrichment for phages
expressing functional scFvs and a corresponding reduction in background (Kristensen &
10 Winter, Folding & Design 3: 321-328, Jul 9, 1998). Selections were performed using
immunotubes coated with either HSA or p-gal at 100p,g/ml concentration.
To check for binding, 24 colonies from the third round of each selection were screened by
monoclonal phage ELISA. Phage particles were produced as described by Harrison et al. 9
15 Methods Enzymol. 1996;267:83-109. 96-well ELISA plates were coated with lOOjul of
HSA or P-gal at 10|j,g/ml concentration in PBS overnight at 4°C. A standard ELISA
protocol was followed (Hoogenboom et al. 9 1991) using detection of bound phage with
anti-M13-HRP conjugate. A selection of clones gave ELISA signals of greater than 1.0
with 50|al supernatant.
20
Next, DNA preps were made from V H /dummy V K library selected on HSA and from
V K /dummy V H library selected on p-gal using the QIAprep Spin Miniprep kit (Qiagen).
To access most of the diversity, DNA preps were made from each of the three rounds of
selections and then pulled together for each of the antigens. DNA preps were then
25 digested with Sali/NotT overnight at 37°C. Following gel purification of the fragments,
V K chains from the V K /dummy V H library selected on P-gal were ligated in place of a
dummy V K chain of the V H /dummy V K library selected on HSA creating a library of 3.3
x 10^ clones.
30 This library was then either selected on HSA (first round) and p-gal (second round),
HSA/p-gal selection, or on p-gal (first round) and HSA (second round), P-gal/HSA
WO 2004/003019 PCT/GB2003/002804
87
selection. Selections were performed as described above. In each case after the second
round 48 clones were tested for binding to HSA and P-gal by the monoclonal phage
ELISA (as described above) and by ELISA of the soluble scFv fragments. Soluble
antibody fragments were produced as described by Harrison et aL, (1996), and standard
5 ELISA protocol was followed Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133,
except that 2% Tween/PBS was used as a blocking buffer and bound scFvs were detected
with Protein L-HRP. Three clones (E4, E5 and E8) from the HSA/p-gal selection and two
clones (K8 and K10) from the (3-gal/HSA selection were able to bind both antigens. scFvs
from these clones were PCR amplified and sequenced as described by Ignatovich et aL 9
10 (1999) J Mol Biol 1999 Nov 26;294(2):457-65, using the primers LMB3 and pHENseq.
Sequence analysis revealed that all clones were identical. Therefore, only one clone
encoding a dual specific antibody (K8) was chosen for further work (Figure 3).
15 Example 2. Characterisation of the binding properties of the K8 antibody.
Firstly, the binding properties of the K8 antibody were characterised by the monoclonal
phage ELISA. A 96-well plate was coated with lOOjul of HSA and p-gal alongside with
alkaline phosphatase (APS), bovine serum albumin (BSA), peanut agglutinin, lysozyme
20 and cytochrome c (to check for cross-reactivity) at 10|Lig/ml concentration in PBS
overnight at 4°C. The phagemid from K8 clone was rescued with KM13 as described by
Harrison et al. 9 (1996) and the supernatant (50|lx1) containing phage assayed directly. A
standard ELISA protocol was followed (Hoogenboom et aL 9 1991) using detection of
bound phage with anti-M13-HRP conjugate. The dual specific K8 antibody was found to
25 bind to HSA and p-gal when displayed on the surface of the phage with absorbance
signals greater than 1.0 (Figure 4). Strong binding to BSA was also observed (Figure 4).
Since HSA and BSA are 76% homologous on the amino acid level, it is not surprising
that K8 antibody recognised both of these structurally related proteins. No cross-reactivity
with other proteins was detected (Figure 4).
30
Secondly, the binding properties of the K8 antibody were tested in a soluble scFv ELISA.
Production of the soluble scFv fragment was induced by IPTG as described by Harrison
et al. 9 (1996). To determine the expression levels of K8 scFv, the soluble antibody
WO 2004/003019 PCT/GB2003/002804
88
fragments were purified from the supernatant of 50ml inductions using Protein A-
Sepharose columns as described by Harlow and Lane, Antibodies: a Laboratory Manual,
(1988) Cold Spring Harbor. OD28O was ^en measured and the protein concentration
calculated as described by Sambrook et aL, (1989). K8 scFv was produced in supernatant
5 at 19mg/l.
A soluble scFv ELISA was then performed using known concentrations of the K8
antibody fragment. A 96-well plate was coated with 100|ul of HSA, BSA and p-gal at
lOjag/ml and lOOjul of Protein A at 1 \xg/ml concentration. 50jli1 of the serial dilutions of
10 the K8 scFv was applied and the bound antibody fragments were detected with Protein L-
HRP. ELISA results confirmed the dual specific nature of the K8 antibody (Figure 5).
To confirm that binding to (3-gal is determined by the V K domain and binding to
HSA/BSA by the Vh domain of the K8 scFv antibody, the V K domain was cut out from
15 K8 scFv DNA by Sali/Not/ digestion and ligated into a SalZ/NotT digested pIT2 vector
containing dummy V H chain (Figures 1 and 2). Binding characteristics of the resulting
clone K8V K /dummy Vh were analysed by soluble scFv ELISA. Production of the soluble
scFv fragments was induced by IPTG as described by Harrison et al. 9 (1996) and the
supernatant (50|lx) containing scFvs assayed directly. Soluble scFv ELISA was performed
20 as described in Example 1 and the bound scFvs were detected with Protein L-HRP. The
ELISA results revealed that this clone was still able to bind p-gal, whereas binding to
BSA was abolished (Figure 6).
Example 3. Selection of single Vjj domain antibodies antigens A and B and single
25 V K domain antibodies directed against antigens C and D.
This example describes a method for making single Vjj domain antibodies directed
against antigens A and B and single V K domain antibodies directed against antigens C
and D by selecting repertoires of virgin single antibody variable domains for binding to
30 these antigens in the absence of the complementary variable domains.
WO 2004/003019 PCT/GB2003/002804
89
Selections and characterisation of the binding clones is performed as described
previously (see Example 5, PCT/GB 02/003014). Four clones are chosen for further
work:
VHl-Anti AV H
VH2-AntiB V H
VK1 - Anti C V K
VK2 — Anti D V K
The procedures described above in Examples 1-3 may be used, in a similar manner as that
described, to produce dimer molecules comprising combinations of V H domains (i.e., V H -
Vh ligands) and cominations of Vl domains (V L -V L ligands).
Example 4. Creation and characterisation of the dual specific ScFv antibodies
(VH1/VH2 directed against antigens A and B and VK1 /VK2 directed against
antigens C and D).
This example demonstrates that dual specific ScFv antibodies (VH1/VH2 directed against
antigens A and B and VK1/VK2 directed against antigens C and D) could be created by
combining V K and V H single domains selected against respective antigens in a ScFv
vector.
To create dual specific antibody VH1 /VH2, VH1 single domain is excised from variable
domain vector 1 (Figure 7) by NcoVXhdl digestion and ligated into NcoVXlioI digested
variable domain vector 2 (Figure 7) to create VH1/ variable domain vector 2. VH2 single
domain is PGR amplified from variable domain vector 1 using primers to introduce Sail
restriction site to the 5' end and NotI restriction site to the 3 5 end. The PCR product is
then digested with SaWNotl and ligated into SaWNotl digested VH1/ variable domain
vector 2 to create VH1/VH2/ variable domain vector 2.
VK1/VK2/ variable domain vector 2 is created in a similar way. The dual specific nature
of the produced VH1/VH2 ScFv and VK1/VK2 ScFv is tested in a soluble ScFv ELISA
WO 2004/003019 PCT/GB2003/002804
90
as described previously (see Example 6, PCT/GB 02/003014). Competition ELISA is
performed as described previously (see Example 8, PCT/GB 02/003014).
Possible outcomes:
-VH1/VH2 ScFv is able to bind antigens A and B simultaneously
-VK1/VK2 ScFv is able to bind antigens C and D simultaneously
-VH1/VH2 ScFv binding is competitive (when bound to antigen A, VH1/VH2 ScFv
cannot bind to antigen B)
-VK1/VK2 ScFv binding is competitive (when bound to antigen C, VK1/VK2 ScFv
cannot bind to antigen D)
Example 5. Construction of dual specific VH1/VH2 Fab and VK1/VK2 Fab and
analysis of their binding properties.
To create VH1/VH2 Fab, VH1 single domain is ligated into NcoVXhol digested CH
vector (Figure 8) to create VH1/CH and VH2 single domain is ligated into SaWNotl
digested CK vector (Figure 9) to create VH2/CK. Plasmid DNA from VH1/CH and
VH2/CK is used to co-transform competent E. coli cells as described previously (see
Example 8, PCT/GB02/003014).
The clone containing VH1/CH and VH2/CK plasmids is then induced by IPTG to
produce soluble VH1/VH2 Fab as described previously (see Example 8, PCT/GB
02/003014).
VK1/VK2 Fab is produced in a similar way.
Binding properties of the produced Fabs are tested by competition ELISA as described
previously (see Example 8, PCT/GB 02/003014).
Possible outcomes:
-VH1/VH2 Fab is able to bind antigens A and B simultaneously
-VK1/VK2 Fab is able to bind antigens C and D simultaneously
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-VH1/VH2 Fab binding is competitive (when bound to antigen A, VH1/VH2 Fab cannot
bind to antigen B)
-VK1/VK2 Fab binding is competitive (when bound to antigen C, VK1/VK2 Fab cannot
bind to antigen D)
5
Example 6
Chelating dAb Dimers
10 Summary
VH and VK homo-dimers are created in a dAb-linker-dAb format using flexible
polypeptide linkers. Vectors were created in the dAb linker-dAb format containing
glycine-serine linkers of different lengths 3U:(Gly4Ser) 3 , 5U:(Gly4Ser) 5 , 7U:(Gly4Ser) 7 .
Dimer libraries were created using guiding dAbs upstream of the linker: TAR1-5 (VK),
15 TAR1-27(VK), TAR2-5(VH) or TAR2-6(VK) and a library of corresponding second
dAbs after the linker. Using this method, novel dimeric dAbs were selected. The effect of
dimerisation on antigen binding was determined by ELISA and BIAcore studies and in
cell neutralisation and receptor binding assays. Dimerisation of both TAR1-5 and TAR1-
27 resulted in significant improvement in binding affinity and neutralisation levels.
20
1.0 Methods
1.1 Library generation
1.1.1 Vectors
pEDA3U, pEDA5U and pEDA7U vectors were designed to introduce different linker
25 lengths compatible with the dAb-linker-dAb format. For pEDA3U, sense and anti-sense
73 -base pair oligo linkers were annealed using a slow annealing program (95°C-5mins,
80°C-10mins, 70°C-15mins 5 56°C-15mins ? 42°C until use) in buffer containing
O.lMNaCl, lOmM Tris-HCl pH7.4 and cloned using the XJ10I and Notl restriction sites.
The linkers encompassed 3 (Gly4Ser) units and a staffer region housed between Sail and
30 Notl cloning sites (scheme 1). In order to reduce the possibility of monomelic dAbs
being selected for by phage display, the stuffer region was designed to include 3 stop
codons, a Sacl restriction site and a frame shift mutation to put the region out of frame
when no second dAb was present. For pEDASU and 7U due to the length of the linkers
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required, overlapping oligo-linkers were designed for each vector, annealed and
elongated using Klenow. The fragment was then purified and digested using the
appropriate enzymes before cloning using ths Xhol and Notl restriction sites.
Ncol
Xhol
Linker:
3U
5U
Sail
71)
r
Notl
Stuffer 1
Staffer 2
Scheme 1
1.1.2 Library preparation
The N~terminal V gene corresponding to the guiding dAb was cloned upstream of the
10 linker using Ncol and Xhol restriction sites. VH genes have existing compatible sites,
however cloning VK genes required the introduction of suitable restriction sites. This was
achieved by using modifying PCR primers (VK-DLIBF: 5' cggccatggcgtcaacggacat;
VKXholR: 5' atgtgcgctcgagcgtttgattt 3') in 30 cycles of PCR amplification using a 2:1
mixture of SuperTaq (HTBiotechnology Ltd)and pfu turbo (Stratagene). This maintained
15 the Ncol site at the 5' end while destroying the adjacent Sail site and introduced the
Xhol site at the 3' end. 5 guiding dAbs were cloned into each of the 3 dimer vectors:
TAR1-5 (VK), TAR1-27(VK), TAR2-5(VH), TAR2-6(VK) and TAR2-7(VK). All
constructs were verified by sequence analysis.
20 Having cloned the guiding dAbs upstream of the linker in each of the vectors (pEDA3U,
5U and 7U): TAR1-5 (VK), TAR1-27(VK), TAR2-5(VH) or TAR2-6(VK) a library of
corresponding second dAbs were cloned after the linker. To achieve this, the
complimentary dAb libraries were PCR amplified from phage recovered from round 1
selections of either a VK library against Human TNFoc (at approximately 1 x 10 6 diversity
25 after round 1) when TAR1-5 or TAR1-27 are the guiding dAbs, or a VH or VK library
against human p55 TNP receptor (both at approximately 1 x 10 5 diversity after round 1)
when TAR2-5 or TAR2-6 respectively are the guiding dAbs. For VK libraries PCR
amplification was conducted using primers in 30 cycles of PCR amplification using a 2:1
mixture of SuperTaq and pfu turbo. VH libraries were PCR amplified using primers in
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order to introduce a Sail restriction site at the 5' end of the gene. The dAb library PCRs
were digested with the appropriate restriction enzymes, ligated into the corresponding
vectors down stream of the linker, using Sall/Notl restriction sites and electroporated
into freshly prepared competent TGI cells.
5
The titres achieved for each library are as follows:
TAR1-5: pEDA3U = 4xl0 8 , pEDA5U = 8xl0 7 , pEDA7U = 1x10 s
TAR1-27: pEDA3U = 6.2xl0 8 , pEDA5U = 1x10 s , pEDA7U = lxlO 9
TAR2h-5: pEDA3U = 4xl0 7 , pEDA5U = 2 x 10 8 , pEDA7U = 8xl0 7
10 TAR2h-6: pEDASU = 7.4xl0 8 , pEDASU = 1.2 x 10 8 , pEDA7U - 2.2xl0 8
1.2 Selections
1.2.1 TNFa
Selections were conducted using human TNFa passively coated on immunotubes.
15 Briefly, Immunotubes are coated overnight with 1 -4mls of the required antigen. The
immunotubes were then washed 3 times with PBS and blocked with 2%milk powder in
PBS for l-2hrs and washed a further 3 times with PBS. The phage solution is diluted in
2%milk powder in PBS and incubated at room temperature for 2hrs. The tubes are then
washed with PBS and the phage eluted with lmg/ml trypsin-PBS. Three selection
20 strategies were investigated for the TAR 1-5 dimer libraries. The first round selections
were carried out in immunotubes using human TNFa coated at 1 jag/ml or 20|ug/ml with
20 washes in PBS 0.1%Tween. TGI cells are infected with the eluted phage and the titres
are determined (eg, Marks et al J Mol Biol. 1991 Dec 5;222(3):581-97, Richmann et al
Biochemistry. 1993 Aug 31;32(34):8848-55).
25
The titres recovered were:
pEDA3U = 2.8xl0 7 (l^g/ml TNF) 1.5x10 s (20]Lig/mlTNF),
pEDA5U = 1.8xl0 7 (ljag/ml TNF), 1.6x10 s (20|ag/ml TNF)
pEDA7U = 8xl0 6 (lug/ml TNF), 7xl0 7 (20p,g/ml TNF).
30
The second round selections were carried out using 3 different methods.
1. In immunotubes, 20 washes with overnight incubation followed by a further 10
washes.
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2. In immunotubes, 20 washes followed by lhr incubation at RT in wash buffer
with (ljj,g/ml TNFa) and 10 further washes.
3. Selection on streptavidin beads using 33 pmoles biotinylated human TNFa
(Henderikx et aL, 2002, Selection of antibodies against biotinylated antigens.
Antibody Phage Display : Methods and protocols, Ed. O'Brien and Atkin, Humana
Press). Single clones from round 2 selections were picked into 96 well plates and
crude supernatant preps were made in 2ml 96 well plate format.
Round 1
Round 2
Round 2
Round 2
Human
selection
selection
selection
TNFaimmuno
method 1
method 2
method 3
tube coating
concentration
pEDA3U
lu.g/ml
1 x 10 y
1.8 x 10 y
2.4 x 10 1U
pEDA3U
20(j.g/ml
6x 10 y
1.8 x 10 10
8.5 x 10 iO
pEDA5U
l|j,g/ml
9x 10*
1.4 x 10 y
2.8 x 10 10
pEDA5U
20|j,g/ml
9.5 x 10 y
8.5 x 10 9
2.8 x 10 10
pEDA7U
lu-g/ml
7.8 x 10 s
1.6 x 10*
4x 10 1U
pEDA7U
20u.g/ml
1 x 10 1U
8x 10 9
1.5 x 10 10
For TAR1-27, selections were carried out as described previously with the following
modifications. The first round selections were carried out in immunotubes using human
TNFa coated at l|Lxg/ml or 20jj.g/ml with 20 washes in PBS 0.1%Tween. The second
round selections were carried out in immunotubes using 20 washes with overnight
incubation followed by a further 20 washes. Single clones from round 2 selections were
picked into 96 well plates and crude supernatant preps were made in 2ml 96 well plate
format.
TAR1-27 titres are as follows:
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T-Ti Tm qti
jn uiiid.il
TTsTF' a 1 Tn m 1 1 n r\ti i V»
X1NJL L^llillll U.11L/LU.UC
coatine cone
IVOUIIU- 1
XvUUllU. Z,
pEDA3U
lug/ml
4x 10 y
6x 10 y
pEDA3U
20 jag/ml
5x 10 y
4.4 x 10 10
pEDA5U
l|ng/ml
1.5 x 10 y
1.9 x 10 i0
pEDA5U
20|iig/ml
3.4 x 10 y
3.5 x 10 10
pEDA7U
l|ag/ml
2.6 x 10 y
5x 10 y
pEDA7U
20p,g/ml
7xlO y
1.4 x 10 i0
1.2.2 TNF RECEPTOR 1 (p55 RECEPTOR; TAR2)
Selections were conducted as described previously for the TAR2h-5 libraries only. 3
rounds of selections were carried out in immunotubes using either l|j.g/ml human p55
5 TNF receptor or 10|Lig/ml human p55 TNF receptor with 20 washes in PBS 0.1%Tween
with overnight incubation followed by a further 20 washes. Single clones from round 2
and 3 selections were picked into 96 well plates and crude supernatant preps were made
in 2ml 96 well plate format.
10 TAR2h-5 titres are as follows:
Round 1 human
p55 TNF
receptor
immunotube
coating
concentration
Round 1
Round 2
Round 3
pEDA3U
l[j.g/ml
2.4 x 10 6
1.2 x 10 7
1.9 x 10 y
pEDA3U
10u.g/ml
3.1 x 10 7
7 x 10'
1 x 10 y
pEDA5U
l|j.g/ml
2.5 x 10 &
1.1 x 10 7
5.7 x 10 s
pEDA5U
10u.g/ml
3.7 x 10 7
2.3 x 10 K
2.9 x 10 y
pEDA7U
lM.g/ml
1.3 x 10 6
1.3 x 10'
1.4 x 10 y
pEDA7U
10|j.g/ml
1.6 x 10 7
1.9 x 10 7
3x 10 1U
1.3 Screening
Single clones from round 2 or 3 selections were picked from each of the 3U 5 5U and 7U
15 libraries from the different selections methods, where appropriate. Clones were grown in
2xTY with 100(j,g/ml ampicillin and 1% glucose overnight at 37°C. A 1/100 dilution of
this culture was inoculated into 2mls of 2xTY with lOOjLig/ml ampicillin and 0.1%
glucose in 2ml, 96 well plate format and grown at 37°C shaking until OD600 was
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approximately 0.9. The culture was then induced with ImM IPTG overnight at 30°C.
The supernatants were clarified by centrifugation at 4000rpm for 15 mins in a sorval plate
centrifuge. The supernatant preps the used for initial screening.
1.3.1 ELISA
Binding activity of dimeric recombinant proteins was compared to monomer by Protein
A/L ELISA or by antigen ELISA. Briefly, a 96 well plate is coated with antigen or
Protein A/L overnight at 4°C. The plate washed with 0.05% Tween-PBS, blocked for 2hrs
with 2% Tween-PBS. The sample is added to the plate incubated for 1 hr at room
temperature. The plate is washed and incubated with the secondary reagent for lhr at
room temperature. The plate is washed and developed with TMB substrate. Protein A/L-
HRP or fridia-HRP was used as a secondary reagent. For antigen ELISAs, the antigen
concentrations used were ljug/ml in PBS for Human TNFa and human THF receptor 1.
Due to the presence of the guiding dAb in most cases dimers gave a positive ELISA
signal therefore off rate determination was examined by BIAcore.
1.3.2 BIAcore
BIAcore analysys was conducted for TAR1-5 and TAR2h-5 clones. For screening,
Human TNFotwas coupled to a CMS chip at high density (approximately 10000 RUs).
50 jlxI of Human TNFoc(50 |Lig/ml) was coupled to the chip at 5|al/min in acetate buffer -
pH5.5. Regeneration of the chip following analysis using the standard methods is not
possible due to the instability of Human TNFa, therefore after each sample was analysed,
the chip was washed for 1 Omins with buffer.
For TAR1-5, clones supernatants from the round 2 selection were screened by BIAcore.
48 clones were screened from each of the 3U, 5U and 7U libraries obtained using the
following selection methods:
Rl: lju,g/ml human TNFa immunotube, R2 1 jag/ml human TNFa immunotube, overnight
wash.
Rl: 20/ig/ml human TNFa immunotube, R2 20jng/ml human TNFa immunotube,
overnight wash.
Rl: l/ig/ml human TNFa immunotube, R2 33 pmoles biotinylated human TNFa on
beads.
Rl : 20jtig/ml human TNFa immunotube, R2 33 pmoles biotinylated human TNFa beads.
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For screening, human p55 TNF receptor was coupled to a CM5 chip at high density
(approximately 4000 RUs). 100 jllI of human p55 TNF receptor (10 )ng/ml) was coupled
to the chip at 5pl/min in acetate buffer - pH5.5. Standard regeneration conditions were
5 examined ( glycine pH2 or pH3) but in each case antigen was removed from the surface
of the chip therefore as with TNFa, therefore after each sample was analysed, the chip
was washed for 1 Omins with buffer.
For TAR2-5, clones supernatants from the round 2 selection were screened.
48 clones were screened from each of the 3U, 5U and 7U libraries, using the following
1 0 selection methods :
Rl: 1/xg/ml human p55 TNF receptor immunotube, R2 1/xg/ml human p55 TNF receptor
immunotube, overnight wash.
Rl: 1 0/xg/ml human p55 TNF receptor immunotube, R2 10/xg/ml human p55 TNF
receptor immunotube, overnight wash.
15
1.3.3 Receptor and Cell Assays
The ability of the dimers to neutralise in the receptor assay was conducted as follows:
Receptor binding
20 Anti-TNF dAbs were tested for the ability to inhibit the binding of TNF to recombinant
TNF receptor 1 (p55). Briefly, Maxisorp plates were incubated overnight with 30mg/ml
anti-human Fc mouse monoclonal antibody (Zymed, San Francisco, USA). The wells
were washed with phosphate buffered saline (PBS) containing 0.05% Tween-20 and then
blocked with 1% BSA in PBS before being incubated with lOOng/ml TNF receptor 1 Fc
25 fusion protein (R&D Systems, Minneapolis, USA). Anti-TNF dAb was mixed with TNF
which was added to the washed wells at a final concentration of lOng/ml. TNF binding
was detected with 0.2mg/ml biotinylated anti-TNF antibody (HyCult biotechnology,
Uben, Netherlands) followed by 1 in 500 dilution of horse radish peroxidase labelled
streptavidin (Amersham Biosciences, UK) and then incubation with TMB substrate (KPL,
30 Gaithersburg, USA). The reaction was stopped by the addition of HC1 and the absorb ance
was read at 450nm. Anti-TNF dAb activity lead to a decrease in TNF binding and
therefore a decrease in absorbance compared with the TNF only control.
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L929 Cytotoxicity Assay
Anti-TNF dAbs were also tested for the ability to neutralise the cytotoxic activity of TNF
on mouse L929 fibroblasts (Evans, T. (2000) Molecular Biotechnology 15, 243-248).
Briefly, L929 cells plated in microtitre plates were incubated overnight with anti-TNF
dAb, lOOpg/ml TNF and lmg/ml actinomycin D (Sigma, Poole, UK). Cell viability was
measured by reading absorbance at 490nm following an incubation with [3-(4,5-
dimethylthiazol-2-yl)-5-(3-carbboxymethoxyphenyl)-2-(4-sulfophenyl)-2H
(Promega, Madison, USA). Anti-TNF dAb activity lead to a decrease in TNF cytotoxicity
and therefore an increase in absorbance compared with the TNF only control.
In the initial screen, supernatants prepared for BIAcore analysis, described above, were
also used in the receptor assay. Further analysis of selected dimers was also conducted in
the receptor and cell assays using purified proteins.
HeLa IL-8 assay
Anti-TNFRl or anti-TNF alpha dAbs were tested for the ability to neutralise the
induction of IL-8 secretion by TNF in HeLa cells (method adapted from that of Akeson,
L. et al (1996) Journal of Biological Chemistry 271, 30517-30523, describing the
induction of IL-8 by IL-1 in HUVEC; here we look at induction by human TNF alpha and
we use HeLa cells instead of the HUVEC cell line). Briefly, HeLa cells plated in
microtitre plates were incubated overnight with dAb and 300pg/ml TNF. Post incubation
the supernatant was aspirated off the cells and IL-8 concentration measured via a
sandwich ELISA (R&D Systems). Anti-TNFRl dAb activity lead to a decrease in IL-8
secretion into the supernatant compared with the TNF only control.
The L929 assay is used throughout the following experiments; however, the use of the
HeLa IL-8 assay is preferred to measure anti-TNF receptor 1 (p55) ligands; the presence
of mouse p55 in the L929 assay poses certain limitations in its use.
1.4 Sequence analysis
Dimers that proved to have interesting properties in the BIAcore and the receptor assay
screens were sequenced. Sequences are detailed in the sequence listing.
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1 .5 Formatting
1.5.1 TAR1-5-19 dimers
The TAR1-5 dimers that were shown to have good neutralisation properties were re-
formatted and analysed in the cell and receptor assays. The TAR 1-5 guiding dab was
5 substituted with the affinity matured clone TAR1-5-19. To achieve this TAR 1-5 was
cloned out of the individual dimer pair and substituted with TAR1-5-19 that had been
amplified by PCR. In addition, TAR 1-5- 19 homodimers were also constructed in the 3U,
5U and 7U vectors. The N terminal copy of the gene was amplified by PCR and cloned as
described above and the C-terminal gene fragment was cloned using existing Sail and
10 Notl restriction sites.
1.5.2 Mutagenesis
The amber stop codon present in dAb2, one of the C-terminal dAbs in the TAR 1-5 dimer
pairs was mutated to a glutamine by site-directed mutagenesis.
15
1.5.3 Fabs
The dimers containing TAR 1-5 or TAR1-5-19 were re-formatted into Fab expression
vectors. dAbs were cloned into expression vectors containing either the CK or CH genes
using Sfil and Notl restriction sites and verified by sequence analysis. The CK vector is
20 derived from a pUC based ampicillin resistant vector and the CH vector is derived from a
pACYC chloramphenicol resistant vector. For Fab expression the dAb-CH and dAb-CK
constructs were co-transformed into HB2151 cells and grown in 2xTY containing 0.1%
glucose, 100ju,g/ml ampicillin and lO/jg/ml chloramphenicol.
25 1.5.3 Hinge dimerisation
Dimerisation of dAbs via cystine bond formation was examined. A short sequence of
amino acids EPKS GDKTHTCPP CP a modified form of the human IgGCl hinge was
engineered at the C terminal region on the dAb. An oligo linker encoding for this
sequence was synthesised and annealed, as described previously. The linker was cloned
30 into the pEDA vector containing TAR1-5-19 using Xhol and Notl restriction sites.
Dimerisation occurs in situ in the periplasm.
1.6 Expression and purification
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1.6.1 Expression
Supernatants were prepared in the 2ml, 96-well plate format for the initial screening as
described previously. Following the initial screening process selected dimers were
analysed further. Dirtier constructs were expressed in TOP10F' or HB2151 cells as
supernatants. Briefly, an individual colony from a freshly streaked plate was grown
overnight at 37°C in 2xTY with lOOpg/ml ampicillin and 1% glucose. A 1/100 dilution
of this culture was inoculated into 2xTY with 100p.g/ml ampicillin and 0.1% glucose and
grown at 37°C shaking until OD600 was approximately 0.9. The culture was then induced
with ImM IPTG overnight at 30°C. The cells were removed by centrifugation and the
supernatant purified with protein A or L agarose.
Fab and cysteine hinge dimers were expressed as periplasmic proteins in HB2152 cells.
A 1/100 dilution of an overnight culture was inoculated into 2xTY with 0.1% glucose
and the appropriate antibiotics and grown at 30°C shaking until OD600 was
approximately 0.9. The culture was then induced with ImM IPTG for 3-4 hours at 25°C.
The cells were harvested by centrifugation and the pellet resuspended in periplasmic
preparation buffer (30mM Tris-HCl pH8.0, ImM EDTA, 20% sucrose). Following
centrifugation the supernatant was retained and the pellet resuspended in 5mM MgS04.
The supernatant was harvested again by centrifugation, pooled and purified.
1.6.2 Protein A/L purification
Optimisation of the purification of dimer proteins from Protein L agarose (Affitech,
Norway) or Protein A agarose (Sigma, UK) was examined. Protein was eluted by batch or
by column elution using a peristaltic pump. Three buffers were examined 0.1M
Phosphate-citrate buffer pH2.6, 0.2M Glycine pH2.5 and 0.1M Glycine pH2.5. The
optimal condition was determined to be under peristaltic pump conditions using 0.1M
Glycine pH2.5 over 10 column volumes. Purification from protein A was conducted
peristaltic pump conditions using 0.1M Glycine pH2.5.
1.6.3 FPLC purification
Further purification was carried out by FPLC analysis on the AKTA Explorer 1 00 system
(Amersham Biosciences Ltd). TAR1-5 and TAR1-5-19 dimers were fractionated by
cation exchange chromatography (1ml Resource S — Amersham Biosciences Ltd) eluted
with a 0-1M NaCl gradient in 50mM acetate buffer pH4. Hinge dimers were purified by
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ion exchange (1ml Resource Q Amersham Biosciences Ltd) eluted with a 0-1M NaCl
gradient in 25mMTris HC1 pH 8.0. Fabs were purified by size exclusion chromatography
using a superose 12 (Amersham Biosciences Ltd ) column run at a flow rate of 0.5ml/min
in PBS with 0.05% tween. Following purification samples were concentrated using
vivaspin 5K cut off concentrators (Vivascience Ltd).
2.0 Results
2.1 TAR1-5 dimers
6 x 96 clones were picked from the round 2 selection encompassing all the libraries and
selection conditions. Supernatant preps were made and assayed by antigen and Protein L
ELISA, BIAcore and in the receptor assays. In ELISAs, positive binding clones were
identified from each selection method and were distributed between 3U ? 5U and 7U
libraries. However, as the guiding dAb is always present it was not possible to
discriminate between high and low affinity binders by this method therefore BIAcore
analysis was conducted.
BIAcore analysis was conducted using the 2ml supematants. BIAcore analysis revealed
that the dimer Koff rates were vastly improved compared to monomelic TAR 1-5.
Monomer Koff rate was in the range of 1 0^M compared with dimer Koff rates which
were in the range of 10" 3 - 10~ 4 M. 16 clones that appeared to have very slow off rates
were selected, these came from the 3U, 5U and 7U libraries and were sequenced. In
addition the supematants were analysed for the ability to neutralise human TNFa in the
receptor assay.
6 lead clones (dl-d6 below) that neutralised in these assays and have been sequenced.
The results shows that out of the 6 clones obtained there are only 3 different second dAbs
(dAbl, dAb2 and dAb3) however where the second dAb is found more than once they are
linked with different length linkers:
TARl-5dl: 3U linker 2 nd dAb=dAbl - l|ug/ml Ag immunotube overnight wash
TARl-5d2: 3U linker 2 nd dAb=dAb2 - l^g/ml Ag immunotube overnight wash
TARl-5d3: 5U linker 2 nd dAb=dAb2 - lfig/ml Ag immunotube overnight wash
TARl-5d4: 5U linker 2 nd dAb=dAb3 - 20(ag/ml Ag immunotube overnight wash
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TARl-5d5: 5U linker 2 nd dAb=dAbl - 20jJ,g/ml Ag immunotube overnight wash
TARl-5d6: 7U linker 2 dAb=dAbl- Rl:l|ag/ml Ag immunotube overnight wash,
R2: beads
5 The 6 lead clones were examined further. Protein was produced from the periplasm and
supernatant, purified with protein L agarose and examined in the cell and receptor assays.
The levels of neutralisation were variable (Table 1). The optimal conditions for protein
preparation were determined. Protein produced from HB2151 cells as supernatants gave
the highest yield (approximately lOmgs/L of culture). The supernatants were incubated
10 with protein L agarose for 2hrs at room temperature or overnight at 4°C. The beads were
washed with PBS/NaCl and packed onto an FPLC column using a peristaltic pump. The
beads were washed with 10 column volumes of PBS/NaCl and eluted with 0.1M glycine
pH2.5. In general, dimeric protein is eluted after the monomer.
15 TARl-5dl-6 dimers were purified by FPLC. Three species were obtained, by FPLC
purification and were identified by SDS PAGE. One species corresponds to monomer and
the other two species corresponds to dimers of different sizes. The larger of the two
species is possibly due to the presence of C terminal tags. These proteins were examined
in the receptor assay. The data presented in table 1 represents the optimum results
20 obtained from the two dimeric species (Figure 1 1)
The three second dAbs from the dimer pairs (ie, dAbl, dAb2 and dAb3) were cloned as
monomers and examined by ELISA and in the cell and receptor assay. All three dAbs
bind specifically to TNF by antigen ELISA and do not cross react with plastic or BSA. As
25 monomers, none of the dAbs neutralise in the cell or receptor assays.
2.1.2 TAR1-5-19 dimers
TAR1-5-19 was substituted for TAR1-5 in the 6 lead clones. Analysis of all TAR1-5-19
dimers in the cell and receptor assays was conducted using total protein (protein L
30 purified only) unless otherwise stated (Table 2). TARl-5-19d4 and TARl-5-19d3 have
the best ND50 (~5nM) in the cell assay, this is consistent with the receptor assay results
and is an improvement over TAR1-5-19 monomer (ND 50 ~30nM). Although purified
TAR1-5 dimers give variable results in the receptor and cell assays TAR1-5-19 dimers
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were more consistent. Variability was shown when using different elution buffers during
the protein purification. Elution using 0.1M Phosphate-citrate buffer pH2.6 or 0.2M
Glycine pH2.5 although removing all protein from the protein L agarose in most cases
rendered it less functional.
TARl-5-19d4 was expressed in the fermenter and purified on cation exchange FPLC to
yield a completely pure dimer. As with TARl~5d4 three species were obtained, by FPLC
purification corresponding to monomer and two dimer species. This dimer was amino
acid sequenced. TAR 1-5- 19 monomer and TARl-5-19d4 were then examined in the
receptor assay and the resulting IC50 for monomer was 30nM and for dimer was 8nM.
The results of the receptor assay comparing TAR1-5-19 monomer, TARl-5-19d4 and
TARl-5d4 is shown in figure 10.
TAR1-5-19 homodimers were made in the 3U, 5U and 7U vectors, expressed and purified
on Protein L. The proteins were examined in the cell and receptor assays and the resulting
IC 5 os (for receptor assay) and ND 50 s (for cell assay) were determined (table 3, figure 12).
2.2 Fabs
TAR 1-5 and TAR1-5-19 dimers were also cloned into Fab format, expressed and purified
on protein L agarose. Fabs were assessed in the receptor assays (Table 4). The results
showed that for both TAR1-5-19 and TAR1-5 dimers the neutralisation levels were
similar to the original Gly4Ser linker dimers from which they were derived. A TAR1-5-19
Fab where TAR 1-5- 19 was displayed on both CH and CK was expressed, protein L
purified and assessed in the receptor assay. The resulting IC50 was approximately InM.
2.3 TAR1-27 dimers
3 x 96 clones were picked from the round 2 selection encompassing all the libraries and
selection conditions. 2ml supernatant preps were made for analysis in ELISA and
bioassays. Antigen ELISA gave 71 positive clones. The receptor assay of crude
supernatants yielded 42 clones with inhibitory properties (TNF binding 0-60%). In the
majority of cases inhibitory properties correlated with a strong ELISA signal. 42 clones
WO 2004/003019 PCT/GB2003/002804
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were sequenced, 39 of these have unique second dAb sequences. The 12 dimers that
gave the best inhibitory properties were analysed farther.
The 12 neutralising clones were expressed as 200ml supernatant preps and purified on
5 protein L. These were assessed by protein L and antigen ELISA, BIAcore and in the
receptor assay. Strong positive ELISA signals were obtained in all cases. BIAcore
analysis revealed all clones to have fast on and off rates. The off rates were improved
compared to monomelic TAR1-27, however the off rate of TAR 1-27 dimers was faster
(Koff is approximately in the range of 10" 1 and 10* 2 M) than the TAR1-5 dimers examined
10 previously (Koff is approximately in the range of 10' 3 - 10"* 4 M). The stability of the
purified dimers was questioned and therefore in order to improve stability, the addition on
5%glycerol, 0.5% Triton XI 00 or 0.5% NP40 (Sigma) was included in the purification of
2 TAR1-27 dimers (d2 and dl6). Addition of NP40 or Triton X100™ improved the yield
of purified product approximately 2 fold. Both dimers were assessed in the receptor
15 assay. TARl-27d2 gave IC50 of ~30nM under all purification conditions. TARl-27dl6
showed no neutralisation effect when purified without the use of stabilising agents but
gave an IC50 of ~50nM when purified under stabilising conditions. No further analysis
was conducted.
20 2.4 TAR2-5 dimers
3 x 96 clones were picked from the second round selections encompassing all the libraries
and selection conditions. 2ml supernatant preps were made for analysis. Protein A and
antigen ELISAs were conducted for each plate. 30 interesting clones were identified as
having good off-rates by BIAcore (Koff ranges between 10* 2 - 10" 3 M). The clones were
25 sequenced and 13 unique dimers were identified by sequence analysis.
Table 1: TAR1-5 dimers
Dimer
Cell
type
Purification
Protein
Fraction
Elution
conditions
Receptor/
Cell assay
TARl-5dl
HB2151
Protein L +
FPLC
small dimeric
species
0.1M glycine
pH2.5
RA~30nM
TARl-5d2
HB2151
Protein L +
FPLC
small dimeric
species
0.1M glycine
pH2.5
RA~50nM
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PCT/GB2003/002804
FPLC
species
pH2.5
EL
M
TARl-5d3
HB2151
Protein L +
FPLC
large dimeric
species
0.1M glycine
pH2.5
RA-300
nM
TARl-5d4
HB2151
Protein L +
FPLC
small dimeric
species
0 . 1 M glycine
pH2.5
RA~3n
M
TARl-5d5
HB2151
Protein L +
FPLC
large dimeric
species
0.1M glycine
pH2.5
RA-200
nM
TARl-5d6
HB2151
Protein L
+FPLC
Large dimeric
species
0.1M glycine
pH2.5
RA-100
nM
'•'note dimer 2 and dimer 3 have the same second dAb (called dAb2), however have
different linker lengths (d2 = (Gly 4 Ser) 3 , d3 = (Gly 4 Ser) 3 ). dAbl is the partner dAb to
dimers 1, 5 and 6. dAb3 is the partner dAb to dimer4. None of the partner dAbs
5 neutralise alone. FPLC purification is by cation exchange unless otherwise stated. The
optimal dimeric species for each dimer obtained by FPLC was determined in these
assays.
10 Table 2: TAR1-5-19 dimers
Dimer
Cell type
Purification
Protein
Fraction
Eiution conditions
Recept
or/ Cell
assay
TAR1-5-19 dl
TOPI OF'
Protein L
Total protein
0.1M glycine pH 2.0
RA-15
nM
TAR1-5-19 d2 (no
stop codon)
TOPI OF'
Protein L
Total protein
0.1M glycine pH 2.0 +
0.05%NP40
RA~2n
M
TARl-5-19d3
(no stop codon)
TOPI OF'
Protein L
Total protein
0.1M glycine pH 2.5.+
0.05%NP40
RA~8n
M
TARl-5-19d4
TOPI OF'
Protein L +
FPLC
FPLC purified
fraction
0.1M glycine
pH2.0
RA~2-
5nM
CA-12
nM
TARl-5-19d5
TOPI OF'
Protein L
Total protein
0.1M glycine pH2.0 +
NP40
RA~8n .
M
CA-10
nM
TAR1-5-19 d6
TOPI OF'
Protein L
Total protein
0.1M glycine pH2.0
*
RA-10
nM
Table 3: TAR1-5-19 homodimers
15
Dimer
Cell type
Purification
Protein Fraction
Eiution conditions
Recept
or/ Cell
assay
SUBSTITUTE SHEET (RULE 26)
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106
homodimer
pH2.5
CA-
-3nM
TAR1-5-19 7U
homodimer
HB2151
Protein L
Total protein
0.1M
pH2.5
glycine
RA-
CAS
40nM
45nM
TAR1-5-19 cys
hinge
HB2151
Protein L +
FPLC
FPLC purified
dimer fraction
0.1M
pH2.5
glycine
RA-
-2nM
TAR1-5-
19CH/ TAR1-
5-19 CK
HB2151
Protein
Total protein
0.1M
pH2.5
glycine
RA-
TnM
Table 4:
TAR1-5/TAR1-5-19 Fabs
Dimer
Cell
type
Purification
Protein
Fraction
Elution
conditions
Receptor/ Cell
assay
TAR1-5CH/
JAM /^TZ"
HB2151
Protein L
Total protein
0.1M
pH2.6
citrate
RA
~90nM
TAR1-5CH/
dAb2 CK
HB2151
Protein L
Total protein
0.1M
pH2.5
glycine
RA
CA-
~30nM
~60nM
dAb3CH/
TAR1-5CK
HB2151
Protein L
Total protein
0.1M
pH2.6
citrate
RA-
~100nM
TAR1-5-
19CH/
dAbl CK
HB2151
Protein L
Total protein
0.1M
pH2.0
glycine
RA
-6nM
dAbl CH/
TAR1-5-19CK
HB2151
Protein L
0.1M glycine
pH2.0
Myc/flag
RA-
-6nM
TAR1-5-
19CH/
dAb2 CK
HB2151
Protein L
Total protein
0.1M
pH2.0
glycine
RA-
CA-
-8nM
42nM
TAR1-5-
19CH/
dAb3CK
HB2151
Protein L
Total protein
0.1M
pH2.0
glycine
RA-
-3nM
5
Example 7
dAb dimerisation by terminal cysteine linkage
10 Summary
For dAb dimerisation, a free cysteine has been engineered at the C-terminus of the
protein. When expressed the protein forms a dimer which can be purified by a two step
purification method.
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PCR construction of TAR1-5-19CYS dimer
See example 8 describing the dAb trimer. The trimer protocol gives rise to a mixture of
monomer, dimer and trimer.
5 Expression and purification of TAR1-5-19CYS dimer
The dimer was purified from the supernatant of the culture by capture on Protein L
agarose as outlined in the example 8.
Separation of TAR1-5-19CYS monomer from the TAR1-5-19CYS dimer
10 Prior to cation exchange separation, the mixed monomer/dimer sample was buffer
exchanged into 50 mM sodium acetate buffer pH 4.0 using a PD-10 column (Amersham
Pharmacia), following the manufacturer's guidelines. The sample was then applied to a
lmL Resource S cation exchange column (Amersham Pharmacia), which had been pre-
equilibrated with 50 mM sodium acetate pH 4.0. The monomer and dimer were separated
15 using the following salt gradient in 50 mM sodium acetate pH 4.0:
150 to 200 mM sodium chloride over 15 column volumes
200 to 450 mM sodium chloride over 10 column volumes
450 to 1000 mM sodium chloride over 15 column volumes
20
Fractions containing dimer only were identified using SDS-PAGE and then pooled and
. the pH increased to 8 by the addition of 1/5 volume of 1M Tris pH 8.0.
In vitro functional binding assay: TNF receptor assay and cell assay
25 The affinity of the dimer for human TNFqj was determined using the TNF receptor and
cell assay. IC50 in the receptor assay was approximately 0.3-0.8 nM; ND50 in the cell
assay was approximately 3-8 nM.
Other possible TAR1-5-19CYS dimer formats
30
PEG dimers and custom synthetic maleimide dimers
Nektar (Shearwater) offer a range of bi-maleimide PEGs [mPEG2-(MAL)2 or mPEG-
(MAL)2] which would allow the monomer to be formatted as a dimer, with a small linker
WO 2004/003019
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separating the dAbs and both being linked to a PEG ranging in size from 5 to 40 kDa. It
has been shown that the 5kDa mPEG-(MAL)2 (ie, [TARl-5-19]-Cys-maleimide-PEG x
2, wherein the maleimides are linked together in the dimer) has an affinity in the TNF
receptor assay of - 1-3 nM. Also the dimer can also be produced using TMEA (Tris[2~
maleimidoethyl]amine) (Pierce Biotechnology) or other bi-functional linkers.
It is also possible to produce the disulphide dimer using a chemical coupling procedure
using 2,2'-dithiodipyridine (Sigma Aldrich) and the reduced monomer.
Addition of a polypeptide linker or hinge to the C-terminus of the dAb.
A small linker, either (Gly 4 Ser) n where n=l to 10, eg, 1, 2, 3, 4, 5, 6 or 7, an
immunoglobulin (eg, IgG hinge region or random peptide sequence (eg, selected from a
library of random peptide sequences) can be engineered between the dAb and the terminal
cysteine residue. This can then be used to make dimers as outlined above.
Example 8
dAb trimerisation
Summary
For dAb trimerisation, a free cysteine is required at the C-terminus of the protein. The
cysteine residue, once reduced to give the free thiol, can then be used to specifically
couple the protein to a trimeric maleimide molecule, for example TMEA (Tris[2-
maleimido ethyl] amine) .
PGR construction of TAR1-5-19CYS
The following oligonucleotides were used to specifically PGR TAR1-5-19 with a Sail and
BarnHI sites for cloning and also to introduce a C-terminal cysteine residue:
Sail
Trp Ser Ala Ser Thr Asp* lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val
1 TGG AGC GCG TCG ACG GAC ATC CAG ATG ACC CAG TCT CCA TCC TCT CTG TCT GCA TCT GTA
ACC TCG CGC AGC TGC CTG TAG GTC TAC TGG GTC AGA GGT AGG AGA GAC AGA CGT AGA CAT
Gly Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Ser lie Asp Ser Tyr Leu His Trp
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61 GGA GAC CGT GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC ATT GAT AGT TAT TTA CAT TGG
CCT CTG GCA CAG TGG TAG TGA ACG GCC CGT TCA GTC TCG TAA CTA TCA ATA AAT GTA ACC
Tyr
Gin
Gin
Lys
Pro
Gly
Lys
Ala
Pro
Lys
Leu
Leu
lie
Tyr
Ser
Ala
Ser
Glu
Leu
Gin
121
TAC
CAG
CAG
AAA
CCA
GGG
AAA
GCC
CCT
AAG
CTC
CTG
ATC
TAT
AGT
GCA
TCC
GAG
TTG
CAA
ft TVS
V7 J. V_
XXX
^3\J X
err*
X X
T>7\ f*
J.Ab
Al A
TCA
CGT
AGG
CTC
AAC
GTT
Ser
Gly
Val
Pro
Ser
Arg
Phe
Ser
Gly
Ser
Gly
Ser
Gly
Thr
Asp
Phe
Thr
Leu
Thr
He
181
AGT
GGG
GTC
CCA
TCA
CGT
TTC
AGT
GGC
AGT
GGA
TCT
GGG
ACA
GAT
TTC
ACT
CTC
ACC
ATC
TCA
CCC
CAG
GGT
AGT
GCA
AAG
TCA
CCG
TCA
CCT
AGA
CCC
TGT
CTA
AAG
TGA
GAG
TGG
TAG
Ser
Ser
Leu
Gin
Pro
Glu
Asp
Phe
Ala
Thr
Tyr
Tyr
Cys
Gin
Gin
Val
Val
Trp
Arg
Pro
241
AGC
AGT
CTG
CAA
CCT
GAA
GAT
TTT
GCT
ACG
TAC
TAC
TGT
CAA
CAG
GTT
GTG
TGG
CGT
CCT
TCG
TCA
GAC
GTT
GGA
CTT
CTA
AAA
CGA
TGC
ATG
ATG
ACA
GTT
GTC
CAA
CAC
ACC
GCA
GGA
BamHI
Phe
Thr
Phe
Gly
Gin
Gly
Thr
Lys
Val
Glu
lie
Lys
Arg
Cys
***
* **
~ ~ -w M _
Gly Ser
Gly
301
TTT
ACG
TTC
GGC
CAA
GGG
ACC
AAG
GTG
GAA
ATC
AAA
CGG
TGC
TAA
TAA
GGA
TCC
GGC
AAA
TGC
AAG
CCG
GTT
CCC
TGG
TTC
CAC
CTT
TAG
TTT
GCC
ACG
ATT
ATT
CCT
AGG
CCG
(* start of TAR1-5-19CYS sequence)
Forward primer
5 ' -TGGAGCGCGTC GAC GGAC ATC C AGATGAC C C AGTCTC C A-3 '
Reverse primer
5 '-TTAGCAGCCGGATCCTTATTAGCACCGTTTGATTTCCAC-3 *
The PCR reaction (50\xL volume) was set up as follows: 200jliM dNTPs, 0.4jnM of each
primer, 5 jliL of lOx P/wTurbo buffer (Stratagene), 100 ng of template plasmid (encoding
TAR 1-5- 19), l\xL of PfuTvxbo enzyme (Stratagene) and the volume adjusted to 50|liL
using sterile water. The following PCR conditions were used: initial denaturing step 94
°C for 2 mins, then 25 cycles of 94 °C for 30 sees, 64 °C for 30 sec and 72 °C for 30 sec.
A final extension step was also included of 72 °C for 5 mins. The PCR product was
purified and digested with Sail and BamHI and ligated into the vector which had also
been cut with the same restriction enzymes. Correct clones were verified by DNA
sequencing.
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Expression and purification of TAR1-5-19CYS
TAR1-5-19CYS vector was transformed into BL21 (DE3) pLysS chemically competent
cells (Novagen) following the manufacturer's protocol. Cells carrying the dAb plasmid
were selected for using lOOpg/mL carbenicillin and 37 jig/mL chloramphenicol. Cultures
5 were set up in 2L baffled flasks containing 500 mL of terrific broth (Sigma- Aldrich),
lOOpg/mL carbenicillin and 37 pg/mL chloramphenicol. The cultures were grown at 30
°C at 200rpm to an O.D.600 of 1-1.5 and then induced with ImM EPTG (isopropyl-beta-
D~thiogalactopyranoside, from Melford Laboratories). The expression of the dAb was
allowed to continue for 12-16 hrs at 30 °C. It was found that most of the dAb was present
10 in the culture media. Therefore, the cells were separated from the media by centrifagation
(8,000xg- for 30 mins), and the supernatant used to purify the dAb. Per litre of
supernatant, 30 mL of Protein L agarose (Affitech) was added and the dAb allowed to
batch bind with stirring for 2 hours. The resin was then allowed to settle under gravity for
a further hour before the supernatant was siphoned off The agarose was then packed into
15 a XK 50 column (Amersham Phamacia) and was washed with 10 column volumes of
PBS. The bound dAb was eluted with 100 mM glycine pH 2.0 and protein containing
fractions were then neutralized by the addition of 1/5 volume of 1 M Tris pH 8.0. Per litre
of culture supernatant 20 mg of pure protein was isolated, which contained a 50:50 ratio
of monomer to dimer.
20
Trimerisation of TAR1-5-19CYS
2.5 ml of 100 pM TAR1-5-19CYS was reduce with 5 mM dithiothreitol and left at room
temperature for 20 minutes. The sample was then buffer exchanged using a PD-10
column (Amersham Pharmacia). The column had been pre-equilibrated with 5 mM
25 EDTA, 50 mM sodium phosphate pH 6.5, and the sample applied and eluted following
the manufactures guidelines. The sample was placed on ice until required. TMEA (Tris[2-
maleimidoethyl] amine) was purchased from Pierce Biotechnology. A 20 mM stock
solution of TMEA was made in 100% DMSO (dimethyl sulphoxide). It was found that a
concentration of TMEA greater than 3 : 1 (molar ratio of dAb:TMEA) caused the rapid
30 precipitation and cross-linking of the protein. Also the rate of precipitation and cross-
linking was greater as the pH increased. Therefore using 100 pM reduced TAR1-5-
19CYS, 25 pM TMEA was added to trimerise the protein and the reaction allowed to
proceed at room temperature for two hours. It was found that the addition of additives
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such as glycerol or ethylene glycol to 20% (v/v), significantly reduced the precipitation
of the trimer as the coupling reaction proceeded. After coupling, SDS-PAGE analysis
showed the presence of monomer, dimer and trimer in solution.
Purification of the trimeric TAR1-5-19CYS
40 |J,L of 40% glacial acetic acid was added per mL of the TMEA-TARl-5-19cys reaction
to reduce the pH to ~4. The sample was then applied to a lmL Resource S cation
exchange column (Amersham Pharmacia), which had been pre-equilibrated with 50 mM
sodium acetate pH 4.0. The dimer and trimer were partially separated using a salt gradient
of 340 to 450 mM Sodium chloride, 50 mM sodium acetate pH 4.0 over 30 column
volumes. Fractions containing trimer only were identified using SDS-PAGE and then
pooled and the pH increased to 8 by the addition of 1/5 volume of 1M Tris pH 8.0. To
prevent precipitation of the trimer during concentration steps (using 5K cut off Viva spin
concentrators; Vivascience), 10% glycerol was added to the sample.
In vitro functional binding assay: TNF receptor assay and cell assay
The affinity of the trimer for human TNFa was determined using the TNF receptor and
cell assay. IC50 in the receptor assay was 0.3nM; ND50 in the cell assay was in the range
of 3 to lOnM (eg, 3nM).
Other possible TAR1-5-19CYS trimer formats
TAR1-5-19CYS may also be formatted into a trimer using the following reagents:
PEG trimers and custom synthetic maleimide trimers
Nektar (Shearwater) offer a range of multi arm PEGs, which can be chemically modified
at the terminal end of the PEG. Therefore using a PEG trimer with a maleimide functional
group at the end of each arm would allow the trimerisation of the dAb in a manner similar
to that outlined above using TMEA. The PEG may also have the advantage in increasing
the solubility of the trimer thus preventing the problem of aggregation. Thus, one could
produce a dAb trimer in which each dAb has a C-terminal cysteine that is linked to a
maleimide functional group, the maleimide functional groups being linked to a PEG
trimer.
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Addition of a polypeptide linker or hinge to the C-terminus of the dAb
A small linker, either (Gly 4 Ser) n where n= 1 to 10, eg, 1, 2, 3, 4, 5, 6 or 7 , an
immunoglobulin (eg, IgG hinge region or random peptide sequence (eg, selected from a
library of random peptide sequences) could be engineered between the dAb and the
5 terminal cysteine residue. When used to make multimers (eg, dimers or trimers), this
again would introduce a greater degree of flexibility and distance between the individual
monomers, which may improve the binding characteristics to the target, eg a multisubunit
target such as human TNFo;.
10 Example 9.
Selection of a collection of single domain antibodies (dAbs) directed against human
serum albumin (HSA) and mouse serum albumin (MSA).
15 This example explains a method for making a single domain antibody (dAb) directed
against serum albumin. Selection of dAbs against both mouse serum albumin (MSA) and
human serum albumin (HSA) is described. Three human phage display antibody libraries
were used in this experiment, each based on a single human framework for V H (see
Figure 13: sequence of dummy V H based on V3-23/DP47 and JH4b) or Vk (see Figure
20 15: sequence of dummy Vk based on ol2/o2/DPK9 and Jkl) with side chain diversity
encoded by NNK codons incorporated in complementarity determining regions (CDR1,
CDR2 and CDR3).
Library 1 (Vh):
25 Diversity at positions: H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95,
H97, H98.
Library size: 6.2 x 10
Library 2 (V H ):
30 Diversity at positions: H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95,
H97, H98, H99, H100, HlOOa, HlOOb.
Library size: 4.3 x 10
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Library 3 (Vk):
Diversity at positions: L30, L31, L32, L34, L50, L53, L91, L92, L93, L94, L96
Library size: 2 x 1 0 9
5 The V H and Vk libraries have been preselected for binding to generic ligands protein A
and protein L respectively so that the majority of clones in the unselected libraries are
functional. The sizes of the libraries shown above correspond to the sizes after
preselection.
10 Two rounds of selection were performed on serum albumin using each of the libraries
separately. For each selection, antigen was coated on immunotube (nunc) in 4ml of PBS
at a concentration of lOOjLCg/ml. hi the first round of selection, each of the three libraries
was panned separately against HSA (Sigma) and MSA (Sigma). In the second round of
selection, phage from each of the six first round selections was panned against (i) the
15 same antigen again (eg 1 st round MSA, 2 nd round MSA) and (ii) against the reciprocal
antigen (eg 1 st round MSA, 2 nd round HSA) resulting in a total of twelve 2 nd round
selections. In each case, after the second round of selection 48 clones were tested for
binding to HSA and MSA. Soluble dAb fragments were produced as described for scFv
fragments by Harrison et al, Methods Enzymol. 1996;267:83-109 and standard ELISA
20 protocol was followed (Hoogenboom et al (1991) Nucleic Acids Res., 19: 4133) except
that 2% tween PBS was used as a blocking buffer and bound dAbs were detected with
either protein L-HRP (Sigma) (for the Vks) and protein A -HRP (Amersham Pharmacia
Biotech) (for the Vrs).
25 dAbs that gave a signal above background indicating binding to MSA, HSA or both were
tested in ELISA insoluble form for binding to plastic alone but all were specific for serum
albumin. Clones were then sequenced (see table below) revealing that 21 unique dAb
sequences had been identified. The minimum similarity (at the amino acid level) between
the Vk dAb clones selected was 86.25% ((69/80)xl00; the result when all the diversified
30 residues are different, eg clones 24 and 34). The minimum similarity between the V H dAb
clones selected was 94 % ((127/136)xl00).
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Next, the serum albumin binding dAbs were tested for their ability to capture
biotinylated antigen from solution. ELISA protocol (as above) was followed except that
ELIS A plate was coated with 1 jug/ml protein L (for the V/c clones) and 1 /xg/ml protein A
(for the V H clones). Soluble dAb was captured from solution as in the protocol and
5 detection was with biotinylated MSA or HSA and streptavidin HRP. The biotinylated
MSA and HSA had been prepared according to the manufacturer's instructions, with the
aim of achieving an average of 2 biotins per serum albumin molecule. Twenty four
clones were identified that captured biotinylated MSA from solution in the ELISA. Two
of these (clones 2 and 38 below) also captured biotinylated HSA. Next, the dAbs were
10 tested for their ability to bind MSA coated on a CMS biacore chip. Eight clones were
found that bound MSA on the biacore.
dAb fall
XSIUGS
PQTlf 11T*A
A/TO A
JVlbA
biotinvlated
H
•
in
MSA1
or k CDR1
g~% f% /~\
Diacore :
Vk library 3
template
(dummy)
K
XXXLX
XASXLQS
QQXXXXPXT
2, 4, 7, 41,
K
SSYLN
RASPLQS
QQTYSVPPT
38, 54
K
SSYLN
RASPLQS
QQTYRIPPT
46, 47, 52, 56
K
FKSLK
NASYLQS
QQWYWPVT
13, 15
K
YYHLK
KASTLQS
QQVRKVPRT
30, 35
K
RRYLK
QASVLQS
QQGLYPPIT
19,
K
YNWLK
RASSLQS
QQNWIPRT
22,
K
LWHLR
HASLLQS
QQSAVYPKT
23,
K
FRYLA
HASHLQS
QQRLLYPKT
24,
K
FYHLA
PASKLQS
QQRARWPRT
31,
K
IWHLN
RASRLQS
QQVARVPRT
33,
K
YRYLR
KASSLQS
QQYVGYPRT
34,
K
LKYLK
NASHLQS
QQTTYYPIT
53,
K
LRYLR
KASWLQS
QQVLYYPQT
11,
K
LRSLK
AASRLQS
QQWYWPAT
12,
K
FRHLK
AASRLQS
QQVALYPKT
v
17,
K
RKYLR
TASSLQS
QQNLFWPRT
18,
K
RRYLN
AASSLQS
QQMLFYPKT
v
16, 21
K
IKHLK
GASRLQS
QQGARWPQT
25, 26
K
YYHLK
KASTLQS
QQVRKVPRT
27,
K
YKHLK
NASHLQS
QQVGRYPKT
55,
K
FKSLK
NASYLQS
QQWYWPVT
V H library 1
(and 2)
template
(dummy)
H
XXYXXX
XIXXXGXXTXYADSVKG
XXXX (XXXX) FDY
8,10
H
WVYQMD
S I S AFGAKTL YADS VKG
LSGKFDY
36,
H
WSYQMT
SIS S FGS STLYADS VKG
GRDHNYSLFDY
Captures
biotinylated
S all 4 bind
S both bind
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In all cases the frameworks were identical to the frameworks in the corresponding
dummy sequence, with diversity in the CDRs as indicated in the table above.
Of the eight clones that bound MSA on the biacore, two clones that are highly expressed
5 in E. coli (clones MSA16 and MSA26) were chosen for further study (see example 10).
Full nucleotide and amino acid sequences for MSA16 and 26 are given in figure 16.
Example 10.
10 Determination of affinity and serum half-life in mouse of MSA binding dAbs MSA16
and MSA26.
dAbs MSA16 and MSA26 were expressed in the periplasm of E. coli and purified using
batch absorbtion to protein L-agarose affinity resin (Affitech, Norway) followed by
15 elution with glycine at pH 2.2. The purified dAbs were then analysed by inhibition
biacore to determine K<i. Briefly, purified MSA16 and MSA26 were tested to determine
the concentration of dAb required to achieve 200RUs of response on a biacore CM5 chip
coated with a high density of MSA. Once the required concentrations of dAb had been
determined, MSA antigen at a range of concentrations around the expected Kd was
20 premixed with the dAb and incubated overnight. Binding to the MSA coated biacore chip
of dAb in each of the premixes was then measured at a high flow-rate of 30 jul/minute.
The resulting curves were used to create Klotz plots, which gave an estimated Kid of
200nM for MSA16 and 70nM for MSA 26 (Figure 17 A & B).
25 Next, clones MSA16 and MSA26 were cloned into an expression vector with the HA tag
(nucleic acid sequence: TATCCTTATGATGTTCCTGATTATGCA and amino acid
sequence: YPYDVPDYA) and 2-10 mg quantities were expressed in E. coli and purified
from the supernatant with protein L-agarose affinity resin (Affitech, Norway) and eluted
with glycine at pH2.2. Serum half life of the dAbs was determined in mouse. MSA26
30 and MSA16 were dosed as single i.v. injections at approx 1.5mg/kg into CD1 mice.
Analysis of serum levels was by goat anti-HA (Abeam, UK) capture and protein L-HRP
(invitrogen) detection ELISA which was blocked with 4% Marvel. Washing was with
0.05% tween PBS. Standard curves of known concentrations of dAb were set up in the
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presence of Ixmouse serum to ensure comparability with the test samples. Modelling
with a 2 compartment model showed MSA-26 had a tl/2ce of 0.1 6hr, a tl/2/3 of 14.5hr and
an area under the curve (AUC) of 465hr.mg/ml (data not shown) and MSA- 16 had a tl/2a
of 0.98hr, a tl/2/3 of 36.5hr and an AUC of 913hr.mg/ml (figure 18). Both anti-MSA
5 clones had considerably lengthened half life compared with HEL4 (an anti-hen egg white
lysozyme dAb) which had a tl/2a of 0.06hr, and a tl/2/3 of 0.34hr.
Example 11.
10 Creation of V H -V H and V7e- Vk dual specific Fab like fragments
This example describes a method for making Vh- Vh and V/c-V/c dual specifics as Fab
like fragments. Before constructing each of the Fab like fragments described, dAbs that
bind to targets of choice were first selected from dAb libraries similar to those described
15 in example 9. A V H dAb, HEL4, that binds to hen egg lysozyme (Sigma) was isolated
and a second Vh dAb (TAR2h-5) that binds to TNFa receptor (R and D systems) was also
isolated. The sequences of these are given in the sequence listing. A Vk dAb that binds
TNFa (TAR1-5-19) was isolated by selection and affinity maturation and the sequence is
also set forth in the sequence listing. A second Vk dAb (MSA 26) described in example 9
20 whose sequence is in figure 17B was also used in these experiments.
DNA from expression vectors containing the four dAbs described above was digested
with enzymes Sail and NotI to excise the DNA coding for the dAb. A band of the
expected size (300-400bp) was purified by running the digest on an agarose gel and
25 excising the band, followed by gel purification using the Qiagen gel purification kit
(Qiagen, UK). The DNA coding for the dAbs was then inserted into either the Ch or Ck
vectors (Figs 8 and 9) as indicated in the table below.
dAb
Target antigen
dAb V H or
dAb Vk
Inserted into
vector
tag(C
terminal)
Antibiotic
resisitance
HEL4
Hen egg lysozyme
V H
C H
myc
Chloramphenicol j
TAR2-5
TNF receptor
V h
Ck
flag
Ampicillin
TAR1-5-19
TNF a
Vk
C h
myc
Chloramphenicol
MSA 26
Mouse serum albumin
Vk
Ck
flag
Ampicillin
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The V H C h and Vh Ck constructs were cotransformed into HB2151 cells. Separately, the
V/c Ch and Vk Ck constructs were cotransformed into HB2151 cells. Cultures of each of
the cotransformed cell lines were grown overnight (in 2xTy containing 5% glucose,
10jUg/ml chloramphenicol and 100/ig/ml ampicillin to maintain antibiotic selection for
5 both C H and Ck plasmids). The overnight cultures were used to inoculate fresh media
(2xTy, 10/ig/ml chloramphenicol and 100jU,g/ml ampicillin) and grown to OD 0.7-0.9
before induction by the addition of IPTG to express their C H and Ck constructs.
Expressed Fab like fragment was then purified from the periplasm by protein A
purification (for the contransformed V H C H and V H Ck) and MSA affinity resin
10 purification (for the contransformed V/c Ch and Vk Ck).
Vh-Vh dual specific
Expression of the V H Ch and V H Ck dual specific was tested by running the protein on a
gel. The gel was blotted and a band the expected size for the Fab fragment could be
15 detected on the Western blot via both the myc tag and the flag tag, indicating that both the
V H C h and V H Ck parts of the Fab like fragment were present. Next, in order to
determine whether the two halves of the dual specific were present in the same Fab-like
fragment, an ELISA plate was coated overnight at 4°C with 100 /jlI per well of hen egg
lysozyme (HEL) at 3 mg/ml in sodium bicarbonate buffer. The plate was then blocked
20 (as described in example 1) with 2% tween PBS followed by incubation with the V H Ch
/V h Ck dual specific Fab like fragment. Detection of binding of the dual specific to the
HEL was via the non cognate chain using 9el0 (a monoclonal antibody that binds the
myc tag, Roche) and anti mouse IgG-HRP (Amersham Pharmacia Biotech). The signal
for the V H C h /V h Ck dual specific Fab like fragment was 0.154 compared to a
25 background signal of 0.069 for the V H Ck chain expressed alone. This demonstrates that
the Fab like fragment has binding specificity for target antigen.
V K -V K dual specific
After purifying the contransformed V/c C H and Vk Ck dual specific Fab like fragment on
30 an MSA affinity resin, the resulting protein was used to probe an ELISA plate coated with
lptg/ml TNFa and an ELISA plate coated with lO/xg/ml MSA. As predicted, there was
signal above background when detected with protein L-HRP on bot ELISA plates (data
not shown). This indicated that the fraction of protein able to bind to MSA (and therefore
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purified on the MSA affinity column) was also able to bind TNFa in a subsequent
ELISA, confirming the dual specificity of the antibody fragment. This fraction of protein
was then used for two subsequent experiments. Firstly, an ELISA plate coated with
, 1/^g/ml TNFa was probed with dual specific V/c Ch and V/c C/c Fab like fragment and
also with a control TNFa binding dAb at a concentration calculated to give a similar
signal on the ELISA. Both the dual specific and control dAb were used to probe the
ELISA plate in the presence and in the absence of 2mg/ml MSA. The signal in the dual
specific well was reduced by more than 50% but the signal in the dAb well was not
reduced at all (see figure 19a). The same protein was also put into the receptor assay with
and without MSA and competition by MSA was also shown (see figure 19c). This
demonstrates that binding of MSA to the dual specific is competitive with binding to
TNFa.
Example 12.
Creation of a Vk- Vk dual specific cys bonded dual specific with specificity for
mouse serum albumin and TNFa
This example describes a method for making a dual specific antibody fragment specific
for both mouse serum albumin and TNFa by chemical coupling via a disulphide bond.
Both MSA16 (from example 1) and TAR1-5-19 dAbs were recloned into a pET based
vector with a C terminal cysteine and no tags. The two dAbs were expressed at 4-10 mg
levels and purified from the supernatant using protein L-agarose affinity resin (Affitiech,
Norway). The cysteine tagged dAbs were then reduced with dithiothreitol. The TAR1-5-
1 9 dAb was then coupled with dithiodipyridine to block reformation of disulphide bonds
resulting in the formation of PEP 1-5-19 homodimers. The two different dAbs were then
mixed at pH 6.5 to promote disulphide bond formation and the generation of TAR1-5-19,
MSA16 cys bonded heterodimers. This method for producing conjugates of two unlike
proteins was originally described by King et al (King TP, Li Y Kochoumian L
Biochemistry. 1978 vol 17: 1499-506 Preparation of protein conjugates via intermolecular
disulfide bond formation.) Heterodimers were separated from monomeric species by
cation exchange. Separation was confirmed by the presence of a band of the expected
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size on a SDS geL The resulting heterodimeric species was tested in the TNF receptor
assay and found to have an IC50 for neutralising TNF of approximately 1 8 nM. Next,
the receptor assay was repeated with a constant concentration of heterodimer (18nM) and
a dilution series of MSA and HSA. The presence of HSA at a range of concentrations (up
5 to 2 mg/ml) did not cause a reduction in the ability of the dimer to inhibit TNFa .
However, the addition of MSA caused a dose dependant reduction in the ability of the
dimer to inhibit TNFa (figure 20).This demonstrates that MSA and TNFa compete for
binding to the cys bonded TAR1-5-19, MSA16 dimer.
10 Data Summary
A summary of data obtained in the experiments set forth in the foregoing examples is set
forth in Annex 4.
All publications mentioned in the present specification, and references cited in said
15 publications, are herein incorporated by reference. Various modifications and variations
of the described methods and system of the invention will be apparent to those skilled in
the art without departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred embodiments, it
should be understood that the invention as claimed should not be unduly limited to such
20 specific embodiments. Indeed, various modifications of the described modes for carrying
out the invention which are obvious to those skilled in molecular biology or related fields
are intended to be within the scope of the following claims.
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Annex 1; polypeptides which enhance half-life in vivo.
Alpha- 1 Glycoprotein (Orosomucoid) (AAG)
Alpha- 1 Antichyromotrypsin (ACT)
Alpha- 1 Antitrypsin (AAT)
Alpha- 1 Microglobulin (Protein HC) (AIM)
Alpha-2 Macroglobulin (A2M)
Antithrombin III (AT III)
Apolipoprotein A-l (Apo A-l)
Apoliprotein B (Apo B)
Beta-2-microglobulin (B2M)
Ceruloplasmin (Cp)
Complement Component (C3)
Complement Component (C4)
CI Esterase Inhibitor (CI INH)
C-Reactive Protein (CRP)
Cystatin C (Cys C)
Ferritin (FER)
Fibrinogen (FIB)
Fibronectin (FN)
Haptoglobin (Hp)
Hemopexin (HPX)
Immunoglobulin A (IgA)
Immunoglobulin D (IgD)
Immunoglobulin E (IgE)
Immunoglobulin G (IgG)
Immunoglobulin M (IgM)
Immunoglobulin Light Chains (kapa/lambda)
Lipoprotein(a) [Lp(a)]
Mannose-bindign protein (MBP)
Myoglobin (Myo)
Plasminogen (PSM)
Prealbumin (Transthyretin) (PAL)
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Retinol-binding protein (RBP)
Rheomatoid Factor (RF)
Serum Amyloid A (SAA)
Soluble Tranferrin Receptor (sTfR)
5 Transferrin (Tf)
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Annex 2
Pairing
Therapeutic relevant references.
TNF
ALPHA/TGF-P
• TGF-b and TNF when injected into the ankle joint of collagen induced
arthritis model significantly enhanced joint inflammation. In non-collagen
challenged mice there was no effect.
TNF ALPHA/IL-
1
• TNF and IL-1 synergize in the pathology of uveitis.
• TNF and IL-1 synergize in the pathology of malaria (hypoglycaemia, NO).
• TNF and IL-1 synergize in the induction of polymorphonuclear (PMN)
cells migration in inflammation.
• IL-1 and TNF synergize to induce PMN infiltration into the peritoneum.
• IL-1 and TNF synergize to induce the secretion of IL-1 by endothelial cells.
Important in inflammation.
• IL-1 or TNF alone induced some cellular infiltration into knee synovium.
IL-1 induced PMNs, TNF — monocytes, together tney maucea a more
severe infiltration due to increased PMNs.
• Circulating myocardial depressant substance (present in sepsis) is low
levels of IL-1 and TNFacting synergistically.
TNF ALPHA/IL-2
• Most relating to synergistic activation of killer T-cells.
TNF ALPHA/IL-3
• Synergy of interleukin 3 and tumor necrosis factor alpha in stimulating
clonal growth of acute myelogenous leukemia blasts is the result or
induction of secondary hematopoietic cytokines by tumor necrosis factor
alpha.
• Cancer Res. 1992 Apr 15;52(8):2197-201.
TNF ALPHA/IL-4
• IL-4 and TNF synergize to induce VCAM expression on endothelial cells.
Implied to have a role in asthma. Same for synovium - implicated in RA.
• TNF and IL-4 synergize to mduce 1L-6 expression m Keratrnocytes.
• Sustained elevated levels of VCAM- 1 in cultured fibroblast-like
synoviocytes can be achieved by TNF-alpha in combination with either IL-
4 or IL-13 through increased rriRNA stability. Am J Pathol. 1999
Apr;154(4):l 149-58
TNF ALPHA/IL-5
• Relationship between the tumor necrosis factor system and the serum
mterleukm-4, mterleuKin-3, inter leuKm- o , eosrnopnii canonic protein, ana
immunoglobulin Jb levels in tne uroncniai nypcrreaciiviLy ui duun- oiiu
their children. Allergy Asthma Proc. 2003 Mar-Apr ;24(2): 11 1-8.
rr-vv T"|-» A T* T>TT A /TT /T
TNF ALPHA/IL-o
• 1JN-T and. lJL»-o are potent growtn iactors ior un-z, <x uuvci xiu.iiia.ii mycioma
cell line. Eur J Haematol 1994 Jul;53(l):3 1-7.
TNF ALPHA/IL-8
• TNF and IL-8 synergized with PMNs to activate platelets. Implicated in
Acute Respiratory .Distress oynarome.
• See IL-5/TNF (asthma). Synergism between interleukin-8 and tumor
necrosis factor-alnha for neutrophil-mediated platelet activation. Eur
Cytokine Netw. 1994 Sep-Oct;5(5):455-60. (adult respiratory distress
syndrome (ARDS))
TNF ALPHA/IL-9
TNF ALPHA/IL-
10
• IL-10 induces and synergizes with TNF in the induction of HIV expression
in chronically infected T-cells.
TNF ALPHA/IL-
11
• Cytokines synergistically induce osteoclast differentiation: support by
immortalized or normal calvarial cells. Am J Physiol Cell Physiol 2002
Sep;283(3):C679-87. (Bone loss)
TNF ALPHA/IL-
12
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TNF ALPHA/IL-
13
• Sustained elevated levels of VCAM-1 in cultured fibroblast-like
synoviocytes can be achieved by TNF-alpha in combination with either IL-
4 or IL-13 through increased miviN A. staoiiity. Am J Jratnoi.
Apr; 154(4): 1149-58.
• Interleulcin- 1 i and tumour necrosis iactor-arpna synergisuoaiiy lnouce
eotaxin production in human nasal fibroblasts. Clin Exp Allergy. 2000
iviar,ou^j jj'to-jj.
• Interleukin-13 and tumour necrosis factor-alpha synergistically induce
eotaxin production in human nasal fibroblasts. Clin Exp Allergy. 2000
Mar;30(3):348-55 (allergic inflammation)
• Implications of serum TNF-beta and IL-13 in the treatment response of
childhood nephrotic syndrome. Cytokine. 2003 Feb 7;21(3): 155-9.
TNF ALPHA/IL-
14
• Effects of inhaled tumour necrosis factor alpha in subjects with mild
asthma. Thorax. 2002 Sep;57(9):774-8.
TNF ALPHA/IL-
15
• Effects of inhaled tumour necrosis factor alpha m subjects witn mild
asthma. Thorax. 2002 Sep;57(9):774-8.
TNF ALPHA/IL-
16
• Tumor necrosis factor- alpha-induced synthesis of interleukin-16 in airway
epithelial cells: priming for serotonin stimulation. Am J Respir Cell Mo I
Biol. 2003 Mar;28(3):354-62. (airway inflammation)
• Correlation of circulating interleukin 16 with proinflammatory cytokines in
patients with rheumatoid arthritis. Rheumatology (Oxford). 2001
Apr;40(4):474-5. No abstract available.
• Interleukin 1 6 is up-regulated in Crohn's disease and participates in TNBS
colitis in mice. Gastroenterology. 2000 Oct;119(4):972-82.
TNF ALPHA/IL-
17
• Inhibition of interleukin- 17 prevents the development of arthritis in
vaccinated mice challenged with Borrelia burgdorferi. Infect Immun. 2003
Jun;71(6):3437-42.
• Interleukin 17 synergises with tumour necrosis factor alpha to induce
cartilage destruction in vitro. Ann Rheum Dis. 2002 Oct;61(10):870-6.
• A role of GM-CSF in the accumulation of neutrophils in the airways caused
by IL-17 and TNF-alpha. Eur Respir J. 2003 Mar;21(3):387-93. (Airway
inflammation)
• Abstract Interleukin- 1, tumor necrosis factor alpha, and interleukin- 17
synergistically up-regulate mtnc oxide and prostaglandin E2 production m
explants of human osteoarthritic knee menisci. Arthritis Rheum. 2001
Sep;44(9):2078-83.
TNF ALPHA/IL-
18
• Association of mterleukm-18 expression with enhanced levels or botti
interleukin-lbeta and tumor necrosis factor alpha in knee synovial tissue of
patients witn rneumatoia artnritis. /Lrinriiis Kneum. zuuj reD } **o^zj.jji/-
47.
• Abstract Elevated levels of interleukin- 1 8 and tumor necrosis factor-alpha
m serum 01 patients witn type z Qiaoetes meintus. reiationsnip wiui aiaueuc
nephropathy. Metabolism. 2003 May;52(5): 605-8.
TNF ALPHA/IL-
19
• Abstract IL-19 induces production of IL-6 and TNF-alpha and results in
cell apoptosis through TNF-alpha. J Immunol. 2002 Oct 15;169(8):4288-
97.
TNF ALPHA/IL-
20
• Abstract Cytokines: IL-20 - a new effector in skin inflammation. Curr Biol.
2001 Jul 10;11(13):R53 1-4
TNF
ALPHA/ Complem
ent
• Inflammation and coagulation: implications for the septic patient. Clin
Infect Dis. 2003 May 15;36(10): 1259-65. Epub 2003 May 08. Review.
TNF
ALPHA/IFN-y
• MHC induction in the brain.
• Synergize in anti-viral response/IFN-p induction.
• Neutrophil activation/ respiratory burst.
• Endothelial cell activation
• Toxicities noted when patients treated with TNF/IFN-y as anti-viral therapy
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• Fractalkine expression by human astrocytes.
• Many papers on inflammatory responses - i.e. LPS, also macrophage
activation.
• Anti-TNF and anti-IFN-y synergize to protect mice from lethal
endotoxemia.
TGF-p/IL-1
• Prostaglndin synthesis by osteoblasts
• IL-6 production by intestinal epithelial cells (mflarnmation model)
• Stimulates IL-1 1 and IL-6 in lung fibroblasts (inflammation model)
• IL-6 and IL-8 production in the retina
TGF-p/IL-6
• Chondrocarcoma proliferation
IL-l/IL-2
• B-cell activation
• LAK cell activation
• T-cell activation
• IL-1 synergy with IL-2 in the generation of lymphokine activated killer
p^IIq i<2 mprliatpfl V>v TNF-alnha and beta ( 1 vrnnhotoxin^ . Cytokine. 1992
Nov4(6V479-87
TT 1 /TT 'X
IL-l/IL-4
• B-cell activation
• IL-4 induces IL-1 expression in endothelial cell activation.
TT 1 /TT C
IL-l/IL-5
IL-l/IL-6
• B cell activation
• T cell activation (can replace accessory cells)
• IL-1 induces IL-o expression
• C3 and serum amyloid expression (acute phase response)
• HIV expression
• Cartilage collagen breakdown.
IL-l/IL-7
• IL-7 is requisite for IL-1 -induced thymocyte proliferation. Involvement of
IL-7 in the synergistic effects of granulocyte-macrophage colony-
stimulating factor or tumor necrosis factor with IL-1. J Immunol. 1992 Jan
1;148(1):99-105.
IL-l/IL-8
IL-l/IL-10
IL-l/IL-11
• Cytokines synergistically induce osteoclast differentiation: support by
immortalized or normal calvarial cells. Am J Physiol Cell Physiol 2002
Sep;283(3):C679-87. (Bone loss)
IL-l/IL-16
• Correlation of circulatmg mterleukm 16 with promflammatory cytokmes in
patients with rheumatoid arthritis. Rheumatology (Oxford). 2001
Apr;40(4):474~5. No abstract available.
TT 1 /TT 1 7
• TnTiih-itinn n-f inter1f»itlrin-1 7 nTPvent*? the development of arthritis in
vaccinated mice challenged with Borrelia burgdorferi. Infect Immun. 2003
Jun;71(6):3437-42.
• Contribution of interleukin 17 to human cartilage degradation and synovial
inflammation in osteoarthritis. Osteoarthritis Cartilage. 2002
Oct;10(10):799-807.
• Abstract Interleukin- 1, tumor necrosis factor alpha, and interleukin- 17
synergistically up-regulate nitric oxide and prostaglandin E2 production in
explants of human osteoarthritic knee menisci. Arthritis Rheum. 2001
Sep;44(9):2078-83.
IL-l/IL-18
• Association of interleukin- 1 8 expression with enhanced levels of both
mterleukin-lbeta and tumor necrosis factor alpha in knee synovial tissue of
patients with rheumatoid arthritis. Arthritis Rheum. 2003 Feb;48(2):339-47.
IL-l/IFN-g
IL-2/IL-3
• T-cell proliferation
• B cell proliferation
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IL-2/IL-4
• B-cell proliferation
• T-cell proliferation
• (selectively inducing activation of CD 8 and NK lymphocytes)IL-2R beta
agonist Pl-30 acts in synergy with IL-2, IL-4, IL-9, and IL-15: biological
and molecular effects. J Immunol 2000 Oct 15:165(8 1: 4312-8.
IL-2/IL-5
• B-cell proliferation/ Ig secretion
• IL-5 induces IL-2 receptors on B-cells
IL-2/IL-6
• Development of cvtotoxic T- cells
IL-2/IL-7
IL-2/IL-9
• See IL-2/IL-4 (NK-cells)
IL-2/IL-10
• B-cell activation
IL-2/IL-12
• x.l-iz synergizes witn xi^-z to rnauce lympnoKane-actrvatea cytotoxicity
and perforin and granzyme gene expression in fresh human NK cells. Cell
Immunol 1995 Oct l;165(l):33-43. (T-cell activation)
IL-2/IL-15
• See IL-2/IL-4 (NK cells)
• (l cell activation and proliferation) IL-15 and IL-2: a matter of life and
death for T cells in vivo. Nat Med. 2001 Jan;7(l): 114-8.
IL-2/IL-16
• Synergistic activation of CD4+ T cells by IL-16 and IL-2. J Immunol 1998
JVlar I;loU(3):21 15-20.
IL-2/IL-17
• Evidence for the early involvement of interleukin 17 in human and
experimental renal aiiogratt rejection. J JratnoL z002 Jul;197(3):322-32.
IL-2/IL-18
• interieuKin lo (ii^-io; in synergy with JLL~z induces lethal lung injury m
mice: a potential role for cytokines, chemokines, and natural killer cells in
the pathogenesis of interstitial pneumonia. Blood. 2002 Feb 15;99(4):1289-
98.
IL-2/TGF-R
XJ— / X VJX kJ
» v^uiiLiui ox eiiector rate, iransiorming growtn tactor beta 1 and
nitt^i it/ ujviii z. ayiAcigiZiC lu picvciii dpopiosis and promote eiiector
expansion JExvMed 1995 Ser> 1 -182nV69Q-7no
IL-2/IFN-y
• Ig secretion by B-cells
• IL-2 induces IFN-y expression by T-cells
TT -9/rFTVLrv/ft
• rv one
IL-3/IL-4
• Synergize in mast cell growth
• Synergistic effects of IL-4 and either GM-CSF or IL-3 on the induction of
CD23 expression by human monocytes: regulatory effects of IFN-alpha and
IFN-gamma. Cytokine. 1994 Jul;6(4):407-13.
TT ^ /TT ^
IL-3/IL-6
TT "2 /TT7VT *r
• IL-4 and IFN-ganima synergistically increase total polymeric IgA receptor
levels in human intestinal epithelial cells. Role of protein tyrosine kinases.
J Immunol. 1996 Jun 15;156(12):4807-14.
IL-3/GM-CSF
• Differential regulation of human eosinophil IL-3, IL-5, and GM-CSF
receptor alpna-cnain expression by cytokines: IL-3, IL-5, and GM-CSF
ao wn-reguiate ijl-d receptor alpna expression witn loss oi IL-5
responsiveness, out up-reguiate receptor alpna expression. J Immunol.
2003 Jun l;170(ll):5359-66. (allergic inflammation)
IL-4/IL-2
• IL-4 svnereisticallv enhances both IL-2- and IL-12-indiiceH TFIM- i a p mm ^ \
expression in murine NK cells. Blood. 2003 Mar 13 TEpub ahead of print]
IL-4/IL-5
• Enhanced mast cell histamine etc. secretion in response to IgE
• A Th2-like cytokine response is involved in bullous pemphigoid, the role of
IL-4 and IL-5 in the pathogenesis of the disease. Int J Immunopathol
Pharmacol 1999 May-Aug;12(2):55-61.
IL-4/IL-6
IL-4/IL-10
IL-4/IL-11
• Synergistic interactions between interleukin-1 1 and interlenkin-4 in support
of proliferation of primitive hematopoietic progenitors of mice. Blood.
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1991 Sep 15;78(6):1448-51.
IL-4/IL-12
XI t VI i-J — ' X
• S»vnprai«;tic effects of TL-4 and IL-18 on IL-12-deoendent IFN-eamma
production by dendritic cells. J Immunol 2000 Jan 1;164(1):64-71.
(increase Thl/Th2 differentiation)
• TL-4 ^vnereisticallv enhances both IL-2- and IL-12-induced IFN-I gamma)
expression in murine NK cells. Blood. 2003 Mar 13 fEpub ahead of print!
IL-4/IL-13
• Abstract Interleukin-4 and interleukin- 13 signaling connections maps.
Sripnrr 200*3 Tun 6-300C5625V 1527-8 fallerev asthma")
• Inhibition of the IL-4/IL-13 receptor system prevents allergic sensitization
without affectincr established allerev in a mouse model for allergic asthma.
J Allergy Clin Immunol. 2003 Jun;lll(6):1361-1369.
IL-4/IL-16
• (asthma) Interleukin (IL)-4/IL-9 and exogenous IL-16 induce IL-16
production by BEAS-2B cells, a bronchial epithelial cell line. Cell
Immunol. 2001 Feb l;207(2):75-80
IL-4/IL-17
• Interleukin (IL)-4 and IL-17 synergistically stimulate IL-6 secretion in
liiiman nrtlrvnir* mvnfi Krn i%1 a <;tQ ftif 7 A/fnJ A/fpH 900'? Nnvl 0f5V6^ 1 -4
UU.llJ.Cli.JL V^vJlvllllV/ 111 V Ulllsl vJ UlClO ID . XftL iv JVJ.L/1 JriCU. Z-W l'lUVjXVlJ y.Ww' 1 t .
(Gut inflairimation)
TT A/TT 94
a TT OA ic a-vTAVf^c c/=>r1 V»A/ r rat unr? fnimnn mo r»TT\T^Vl n Cf^c TtYHYilltl (thi rhCr\) 0000
• J.J_/ — Z-T" xo CA.piCbbCLl Uy LcLV cilXKX HU.llla.ll lllav/1 wjJlldgt'S. xfilffllAIl\JlJl>Vl\jx< i y. £*\J\J
Jul;205(3):321-34.
TT 4/TT -0*i
« AKc+rcn-t TsTrtx/' TT _17 fnmilv mpmliprQ nrnmntp TVi! nt Th'? TPWrni^p*? in the
• ADa IXdv/L 1NCW 1J—/ 1 / ldlllliy lliciliut/i o ljx VJ ixivJ LV/ jlj.ii \jx nix ivvojjvJxxot/O xia nx^
lung: in vivo function of the novel cytokine IL-25. J Immunol. 2002 Jul
l;169(l):443-53. (allergic inflammation)
• Abstract Mast cells produce interleukin-25 upon Fcepsilon Rl-mediated
activation. Blood. 2003 May l;101(9):3594-6. Epub 2003 Jan 02. (allergic
1 Tl fl £» TY1TY1 54 "H C\Yl 1
IJLlllallllllaLlAJll y
TT /I /TT7XT
IL-4/lFN-y
• ADSiract inTeneuKin *» niQuces micricuKin o protiuuLiun oy ciiuuuiciiai uciio.
synergy with interferon-gamma. Eur J Immunol. 1 99 1 Jan;2 1 ( 1 ) : 97- 101.
IL-4/SCF
• Regulation of human intestinal mast cells by stem cell factor and IL-4.
Immunol jctev. zuui rcu,i / y.D i -ou. xveview.
IL-5/IL-3
• Differential regulation of human eosinophil IL-3 5 IL-5, and GM-CSF
receptor alpha-chain expression by cytokines: IL-3, IL-5, and GM-CSF
A r\-»im rprm1(j+p TT ^ T"<=»r**=»-r\fr»T cilnrm pynrpccinti ■\x/it"Ti IncG r\~f TT
CIO WIl-lCgUlclLC 11j J ICv/CpiUl dlJJlla CA.piCoolvJll W1LUL IvJoo KJX Xl—i~*J
responsiveness, but up-regulate IL-3 receptor alpha expression. J Immunol.
2003 Jun l;170(ll):5359-66. (Allergic inflammation see abstract)
IL-5/IL-6
IL-5/IL-13
• Inhibition of allergic airways inflammation and airway
hyperresponsiveness in mice by dexamethasone: role of eosinophils, IL-5,
entaxin andTL-13 J Allergy Clin Immunol 2003 Mav*lll(5V 1049-61.
TT -S/TT -1 7
XX-i~^J! XX-J X 1
• TntprlenVin-1 7 nrehe^trates the eranulocvte influx into airwavs after
allergen inhalation in a mouse model of allergic asthma. Am J Respir Cell
MolBiol. 2003 Jan;28(l):42-50.
IL-5/IL-25
• Abstract New IL-17 family members promote Thl or Th2 responses in the
lung: in vivo function of the novel cytokine IL-25. J Immunol. 2002 Jul
1 *1 M(\ \ -44*3 -5 3 (alleroic inflarnmation^
m ATncfrjmt A/fnct pplk •nrndnre inter1enVin-'75 linmi Fcen<iilnn RT-mediated
• ^j.L?OLld^/L lVlCXO L V^-Cllo L/l vJLXLX^/t' JJLXLWJ.XwLlXVJ.XX a<J U.JL/UXX X vwUOXlUll XVI. lUvUlUlUU
activation Blood 2003 Mav 1-101C9V3594-6 Eoub 2003 Jan 02. (allergic
inflammation)
IL-5/IFN-y
IL-5/GM-CSF
• Differential regulation of human eosinophil IL-3, IL-5, and GM-CSF
receptor alpha-chain expression by cytokines: IL-3, IL-5, and GM-CSF
down-regulate IL-5 receptor alpha expression with loss of IL-5
responsiveness, but up-regulate IL-3 receptor alpha expression. J Immunol.
2003 Jun l;170(ll):5359-66. (Allergic inflammation)
IL-6/IL-10
IL-6/IL-11
IL-6/IL-16
• Interleukin-16 stimulates the expression and production of pro-
WO 2004/003019
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127
inflammatory cytokines by human monocytes. Immunology. 2000
IL-6/IL-17
• Stimulation of airway mucin gene expression by interleukin (IL)-17
fhrmio-Ti TT f\ ■nararrmp/aiitocrine loon J Hinl CIipwi 2003 lVfav
9;278(19): 17036-43. Epub 2003 Mar 06. (airway inflammation, asthma)
IL-6/IL-19
• Abstract IL-19 induces production of IL-6 and TNF-alpha and results in
~p.ii nr*n-ntrkcic fhrrmoh TNTF-alrVha / Tmmunnl 900? Oct 1 5-1 69fRV42R8-
97.
TT £/TTh"NJ er
IL-7/IL-2
• Interleukin 7 worsens graft-versus-host disease. Blood. 2002 Oct
l;100(7):2642-9.
EL-7/IL-12
• Synergistic effects of IL-7 and IL-12 on human T cell activation. J
Immunol 1995 May 15;154(10):5093-102.
IL-7/IL-15
• Interleukin-7 and interleukin- 15 regulate the expression of the bcl-2 and c-
myb genes m cutaneous 1-celi lymphoma cells, rSlooa. zuui jnov
l;98(9):2778-83. (growth factor)
IL-8/IL-11
• Abnormal production of interleukin (IL)-1 1 and IL-8 in polycythaemia
vera. Cytokine. 2002 Nov 21 ;20(4): 178-83.
IL-8/IL-17
• The Role of IL-17 in Joint Destruction. Drug News Per sped. 2002
Jan;15(l): 17-23. (arthritis)
• Abstract Interleukin- 17 stimulates the expression of interleukin- 8, growth-
related oncogene-alpha, and granulocyte-colony-stimulating factor by
human airway epithelial cells. Am J Respir Cell Mo I Biol. 2002
Jun;Zo(o): /4o-jo. (airway inriammationj
IL-8/GSF
• Interleukin- 8: an autocrine/paracrine growth factor for human
hematopoietic progenitors acting in synergy with colony stimulating factor-
1 to promote monocyte-macrophage growth and differentiation. Exp
Hematol 1999 Jan;27(l):28-36.
IL-8/VGEF
• Intracavitary VEGF. bFGF, IL-8, IL-12 levels in primary and recurrent
malignant glioma. JNeurooncol. 2003 May;62 (3) .297-303.
IL-9/IL-4
• Anti-mterleukm-9 antibody treatment inhibits airway lntlammation and
hyperreactivity in mouse asthma model. Am J Respir Crit Care Med. 2002
Aug 1;166(3):409-16.
IL-9/IL-5
• Pulmonary overexpression of IL-9 induces Th2 cytokine expression,
leading to immune pathology. J Clin Invest. 2002 Jan;109(l):29-39.
• Th2 cytokines and asthma. Interleukin-9 as a therapeutic target for asthma.
Respir Res. 2001;2(2):o0-4. iipubzUUl reb Id. Keview.
• Abstract mterieuKin-y ennances lnieneuKm- d receptor exprebbion,
differentiation, and survival of human eosinophils. Blood. 2000 Sep
15;96(6):2 163-71 (asthma)
IL-9/IL-13
• Anti-interleukin-9 antibody treatment inhibits airway inflammation and
nyperreacnvity m mouse astnma moaei. yy.7?z »/ itespu i^rii K^are lvieu,
Aug i,ioo(3j.4uy-io.
• Direct effects of interleukin- 13 on epithelial cells cause airway
hyperreactivity and mucus overproduction in asthma. Nat Med. 2002
A.Ug,o^o j.OOJ-7.
IL-9/IL-16
• See IL-4/IL-16
IL-10/IL-2
• The interplay of interleukin- 10 (IL-10) and interleukin-2 (IL-2) in humoral
immune responses: IL-10 synergizes with IL-2 to enhance responses of
human B lymphocytes in a mechanism which is different from upregulation
of CD25 expression. Cell Immunol 1994 Sep;157(2):478-88.
IL-10/IL-12
IL-10/TGF-P
• IL-10 and TGF-beta cooperate in the regulatory T cell response to mucosal
allergens in normal immunity and specific immunotherapy. Eur J
Immunol 2003 May;33(5): 1205-14.
IL-10/IFN-y
WO 2004/003019
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IL-ll/IL-6
• Interleukin-6 and interleukin-1 1 support human osteoclast formation by a
RANKL-mdependent mechanism. Bone, 2003 Jan;32(l):l-7. (bone
resorption in inflammation)
IL-ll/IL-17
• Polarized in vivo expression of IL-1 1 and IL-17 between acute and chronic
skin lesions. J Allergy Clin Immunol 2003 Apr;lll(4):875-81. (allergic
dermatitis i
ytvx Axm nuu r
• IL-1 7 promotes bone erosion in murine collagen-induced arthritis through
loss of the receptor activator of NF-kappa B ligand/osteoprotegerin
balance. J Immunol. 2003 Mar l;170(5):2655-62.
IL-11/TGF-P
• Polarized in vivo expression of IL-1 1 and IL-1 7 between acute and chronic
skin lesions. J Allergy Clin Immunol 2003 Apr;lll(4):875-81. (allergic
dermatitis)
IL-12/IL-13
• Relationship of Interleukin-1 2 and Interleukin-1 3 imbalance with class-
specific rheumatoid factors and anticardiolipin antibodies in systemic lupus
erythematosus. Clin Rheumatol 2003 May;22(2): 107-11.
IL-12/IL-17
• Upregulation of interleukin- 1 2 and - 1 7 in active inflammatory bowel
disease Scand J Gastroenterol 2003 Feb ;38(2): 180-5.
IL-12/IL-18
• Synergistic proliferation and activation of natural killer cells by interleukin
12 and interleukin 18. Cytokine. 1999 Nov;ll(l l):822-30.
• Inflammatory Liver Steatosis Caused bv IL-1 2 and IL-1 8. J Interferon
Cytokine Res. 2003 Mar;23(3): 155-62.
TT -1 ?/TT
• nter1eiikm»23 rather than interleukin- 12 is the critical cvtokine for
autoimmune mflammation of the brain. Nature. 2003 Feb
13;421(6924):744-8.
• Abstract A unique role for IL-23 in promoting cellular immunity. J Leukoc
Biol 2003 Jan;73(l):49-56. Review.
IL-12/IL-27
• Abstract IL-27, a heterodimeric cytokine composed of EBB and p28
protein, induces proliferation of naive CD4(+) T cells. Immunity. 2002
Jun:16f6):779-90.
IL-12/IFN-y
• IL-12 induces IFN-y expression by B and T-cells as part of immune
stimulation.
TT 1 ^/TT
• See TT -5/IL-13
IL-13/IL-25
• Abstract New IL-1 7 family members promote Thl or Th2 responses in the
lung: in vivo function of the novel cytokine IL-25. J Immunol. 2002 Jul
1 •! 69(1 V443-53. (allereic inflammation)
• Abstract Mast cells produce interleukin-25 upon Fcepsilon Rl-mediated
activation. Blood. 2003 May l;101(9):3594-6. Epub 2003 Jan 02. (allergic
inflammation)
IL-15/IL-13
• Differential expression of interleukins (IL)-13 and IL-1 5 in ectopic and
eutopic endometrium of women with endometriosis and normal fertile
women. Am JReprod Immunol. 2003 Feb;49(2):75-83.
IL-15/IL-16
• IL-1 5 and IL-1 6 overexpression in cutaneous T-cell lymphomas: stage-
dependent increase in mycosis fungoides progression. Exp Dermatol. 2000
Aug;9(4):248-51.
IL-15/IL-17
• Abstract IL-1 7, produced by lymphocytes and neutrophils, is necessary for
litDopolvsaccharide-induced airway neutrophilia: IL-1 5 as a possible trigger.
J Immunol 2003 Feb 15;170(4):2106-12. (airway inflammation)
IL-15/IL-21
• IL-2 1 in Synergy with IL-1 5 or IL-1 8 Enhances IFN-gamma Production in
Human NK and T Cells. J Immunol 2003 Jun l;170(ll):5464-9.
IL-17/IL-23
• Interleukin-23 promotes a distinct CD4 T cell activation state characterized
by the production of interleukin- 17. JBiol Chem. 2003 Jan
17;278(3):1910-4. Epub 2002 Nov 03
IL-17/TGF-p
• Polarized in vivo expression of IL-1 1 and IL-1 7 between acute and chronic
skin lesions. J Allergy Clin Immunol 2003 Apr;ll 1(4): 875-81. (allergic
dermatitis)
IL-18/IL-12
• Synergistic proliferation and activation of natural killer cells by interleukin
WO 2004/003019
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129
12 and interleukin 18. Cytokine. 1999 Nov;ll(l l):822-30.
• Abstract Inhibition of in vitro immunoglobulin production by IL- 1 2 in
murine chronic graft-vs.-host disease: synergism with IL-18. Eur J
Immunol 1998 Jun;28(6):20 17-24.
IL-18/IL-21
• IL-21 in Synergy with IL-15 or IL-18 Enhances IFN-gamma Production in
Human NK and T Cells. J Immunol 2003 Jun 1;170(1 1):5464-9.
IL-18/TGF-p
• Interleukin 1 8 and transforming growth factor betal in the serum of
patients with Graves' ophthalmopathy treated with corticosteroids. Int
Immunopharmacol 2003 Apr;3(4): 549-52.
IL-18/IFN-Y
Anti-TNF
ALPHA/anti-CD4
• Synergistic therapeutic effect in DBA/1 arthritic mice.
WO 2004/003019 PCT/GB2003/002804
130
Annex 3: Oncology combinations
Target
Disease
Pair with
CD89*
Use as cytotoxic cell recruiter
all
CD19
B cell lvnTDhotnas
J-* V/VJi AY A.AJ.Jw'AAVo'AAAtliD
HLA-DR
B cell Ivninhcvnia^
J— ' vUJll A V AllLJAAvrAlAClO
v^xy„?
CD38
A/Til 1 tl^l f» YYY\Te*1i~\YV1Cl
ivmiLipic my civ ilia
XXJ_(/\-X-/XV
CD138
lVTultinle mvplnma
At AtAALAJL/AVs AAA Y \s -LVJ LXiCL
CTnR
V^X-/ J) o
XXX_/xjl. XvXV
CD138
T IITIO" PflTlPPr
X_/UXXg l/dXXOC/X
CEA
CD33
A pnfp Tmvplnrl 1 ^rmr^Vi nm^
*VW UXG lllyCHJVl IJ'IIaJPIaIJIIacI
V^X^O^T
XX JLj/\~ J_J X\-
CD56
Lixnf* cancer
A—/ UAlfi vsUAAV/wA
V^X^ 1 JO
CFA
CEA
Pan carrtnoma
A ClAA v/iiA v^AAAUAAAu-
ivxj-i x icocptoi
VEGF
V J / V — 1 A.
P qt) rairin/ima
X dXX OCXXOiAiUlAACt
ivldx receptor
VFGF
TX rj /"» Q •*•/"• 1 "M <""vt"V1 id
x dii udrcinoijia
iVLbl receptor
A V/ O V^IJ I.AJ 1
IL-13
A <5rliTVi5?/n'i'ilTnfYnflT"i7
■Ti-ij ixiixxci/ jj uxxxiuilaX y
inflamma iinn
AAAAAUAA AAAXCA llvll
TT _S
X_i vJ Id A.JXXI O 1
MDC
TARC
IL-9
lAv 7
EGFR
A_/\ IX AX.
CD40L
IL-25
MCP-l
TGFR
X VJX IJ
IL-4
Asthina
IX/ A «J>
IL-5
A J — l w/
Eo taxing s^
MDC
TARC
TNFc£
TT -Q
EGFR
CD40L
IL-25
MCP-l
TGFP
Eotaxin
Asthma
IL-5
Eotaxin-2
Eotaxin-3
EGFR
cancer 1
HER2/neu
HER3
HER4
HER2
cancer
HER3
WO 2004/003019
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131
TNFR1
RA/Crohn's disease
HER4
IL-1R
IL-6R
TNFa
RA/Crohn's disease
IL-la/0
IL-6
IL-18
ICAM-1
IL-15
IL-17
IL-1R
RA/Crohn's disease
IL-6R
IL-18R
IL-18R
RA/Crohn's disease
IL-6R
WO 2004/003019
132
PCT/GB2003/002804
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WO 2004/003019
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PCT/GB2003/002804
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SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
134
PCT/GB2003/002804
Claims
1 . A dual-specific ligand comprising a first immunoglobulin single variable
domain having a binding specificity to a first epitope or antigen and a second
complementary immunoglobulin single variable domain having a binding activity
to a second epitope or antigen, wherein one or both of said antigens or epitopes
acts to increase the half-life of the ligand in vivo and wherein said first and second
domains lack mutually complementary domains which share the same specificity,
provided that said dual specific ligand does not consist of an anti-HS A Vh domain
and an anti-p galactosidase V K domain.
2. A dual-specific ligand according to claim 1, comprising at least one single
heavy chain variable domain of an antibody and one complementary single light
chain variable domain of an antibody such that the two regions are capable of
associating to form a complementary VH/VL pair.
3 . A dual specific ligand according to claim 2 wherein the Vh and Vl are
provided by an antibody scFv fragment.
4. A dual-specific ligand according to claim 2 wherein the Vh and Vl are
provided by an antibody Fab region.
5. A four chain IgG immunoglobulin ligand comprising a dual specific
ligand of claim 2.
6. A four chain IgG immunoglobulin ligand according to claim 5, wherein
said IgG comprises two dual specific ligands, said dual specific ligands being
identical in their variable domains.
7. A four chain IgG immunoglobulin ligand according to claim 5, wherein
said IgG comprises two dual specific ligands, said dual specific ligands being
different in their variable domains.
8. A ligand comprising a first immunoglobulin variable domain having a first
antigen or epitope binding specificity and a second immunoglobulin variable
domain having a second antigen or epitope binding specificity wherein one or
both of said first and second variable domains bind to an antigen which increases
the half-life of the ligand in vivo, and the variable domains are not complementary
to one another.
9. A ligand according to claim 8 wherein the first and the second
immunoglobulin variable domains are heavy chain variable domains (Vh).
10. A ligand according to claim 8 wherein the first and the second
immunoglobulin variable domains are light chain variable domains (Vl).
11. A ligand according to claim any preceding claim, wherein the first and
second epitopes bind independently, such that the dual specific ligand may
simultaneously bind both the first and second epitopes or antigens.
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
135
PCT/GB2003/002804
12. A ligand according to claim 1 1 ? wherein the dual specific ligand comprises
a first form and a second form in equilibrium in solution, wherein both epitopes or
antigens bind to the first form independently but compete for binding to the
second form.
13. A ligand according to any preceding claim wherein the variable regions
are derived from immunoglobulins directed against said epitopes or antigens.
14. A ligand according to any preceding claim, wherein said first and second
epitopes are present on separate antigens.
15. A ligand according to any one of claims 1 to 1 1 5 wherein said first and
second epitopes are present on the same antigen.
16. A ligand according to any preceding claim comprising a variable domain
that is derived from a repertoire of single antibody domains.
17. A ligand of claim 16 wherein said repertoire is displayed on the surface of
filamentous bacteriophage and wherein the single antibody domains are selected
by binding of the bacteriophage repertoire to antigen.
18. A ligand according to any preceding claim wherein the sequence of at
least one variable domain is modified by mutation or DNA shuffling.
19. A dual-specific ligand according to any preceding claim wherein the
variable regions are non-covalently associated.
20. A dual-specific ligand according to any one of claims 1 to 18 wherein the
variable regions are covalently associated.
21. A dual-specific ligand according to claim 20 wherein the covalent
association is mediated by disulphide bonds.
22. A dAb monomer ligand specific for TNFa, which dissociates from human
TNFa with a dissociation constant (Kd) of 50nM to 20pM, and a K 0 ff rate constant
of SxlO" 1 to lxlO" 7 s** 1 , as determined by surface plasmon resonance.
23. A dAb monomer ligand specific for TNFa according to claim 22, wherein
the dAb is a Vk.
24. A dAb monomer ligand specific for TNF receptor 1 (p55), which
dissociates from human TNF receptor 1 with a dissociation constant (Kd) of 50nM
to 20pM, and a K 0 ff rate constant of 5x1 0" 1 to lxl 0" 7 s" 1 , as determined by surface
plasmon resonance.
25. A dAb monomer ligand according to claim 22 to claim 24, wherein the
monomer neutralises human TNFa or TNF receptor 1 in a standard cell assay
with an ND50 of 500nM to 50pM.
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
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PCT/GB2003/002804
26. A dAb monomer ligand specific for TNF receptor 1 (p55), wherein the
dAb antagonises the activity of the TNF receptor 1 in a standard cell assay with
an ND 50 of <100nM, and at a concentration of <10pM the dAb agonises the
activity of the TNF receptor 1 by <5% in the assay.
27. A dAb monomer ligand specific for serum albumin (SA) which dissociates
from SA with a dissociation constant (K d ) of InM to 500jjM ? as determined by
surface plasmon resonance.
28. A dAb monomer ligand according to claim 27, wherein the monomer
binds SA in a standard ligand binding assay with an IC50 of InM to SOOjjM.
29. A dAb monomer ligand specific for TNFa, wherein the dAb comprises
the amino acid sequence of TAR1-5-19 or a sequence that is at least 80%
homologous thereto.
30. A dAb monomer ligand specific for TNFa, wherein the dAb comprises
the amino acid sequence of TAR1-5 or a sequence that is at least 80%
homologous thereto.
31. A dAb monomer ligand specific for TNFa, wherein the dAb comprises
the amino acid sequence of TAR1-27 or a sequence that is at least 80%
homologous thereto.
32. A dAb monomer ligand specific for TNF receptor 1 ? wherein the dAb
comprises the amino acid sequence of TAR2-10 or a sequence that is at least 80%
homologous thereto.
33. A dAb monomer ligand specific for TNF receptor 1, wherein the dAb
comprises the amino acid sequence of TAR2-10 or a sequence that is at least 90%
homologous thereto.
34. A dAb monomer ligand specific for TNF receptor 1 , wherein the dAb
comprises the amino acid sequence of TAR2-5 or a sequence that is at least 80%
homologous thereto.
35. A dAb monomer ligand specific for TNF receptor 1 , wherein the dAb
comprises the amino acid sequence of TAR2-5 or a sequence that is at least 90%
homologous thereto.
36. A dAb monomer ligand specific for SA, wherein the dAb comprises the
amino acid sequence of MSA-16 or a sequence that is at least 80% homologous
thereto.
37. A dAb monomer ligand specific for SA, wherein the dAb comprises the
amino acid sequence of MSA-26 or a sequence that is at least 80% homologous
thereto.
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
PCT/GB2003/002804
137
38. A dAb monomer according to any one of claims 29 to 37, wherein the
TNFoc, TNF receptor 1 or SA is in human form.
39. A dAb monomer further comprising a terminal Cys residue.
40. A dAb monomer according to any one of claims 29 to 38, further
comprising a terminal Cys residue.
41. A dual specific ligand comprising at least one dAb monomer according to
any one of claims 22 to 40.
42. A dual specific ligand according to claim 41 , which is a dimer.
43. A dual specific ligand according to claim 42, wherein the dimer comprises
anti-human TNF alpha dAb according to any one of claims 22, 23 and 28-30, and
an anti-SA dAb according to any one of claims 26, 27 and 34-36.
44. A dual specific ligand according to claim 42, wherein the dimer is a
homo- or hetero-dimer comprising first and second anti-human TNF alpha dAbs,
each dAb being according to any one of claims 22, 23 and 28-30.
45. A dual specific ligand according to claim 41, which is a trimer.
46. A dual specific ligand according to claim 45, which is a homotrimer
comprising three copies of an anti-human TNF alpha dAb according to any one of
claims 22, 23 and 29-3 1 .
47. A ligand according to any preceding claim, which comprises a universal
framework.
48. A ligand according to claim 47, wherein the universal framework
comprises a Vh framework selected from the group consisting of DP47, DP45 and
DP38; and/or the V L framework is DPK9.
49. A ligand according to any preceding claim which comprises a binding site
for a generic ligand.
50. The ligand of claim 49, wherein the generic ligand binding site is selected
from the group consisting of protein A, protein L and protein G.
51. A ligand according to any preceding claim, wherein the ligand comprises a
variable domain having one or more framework regions comprising an amino acid
sequence that is the same as the amino acid sequence of a corresponding
framework region encoded by a human germline antibody gene segment, or the
amino acid sequences of one or more of said framework regions collectively
comprises up to 5 amino acid differences relative to the amino acid sequence of
said corresponding framework region encoded by a human germline antibody
gene segment.
SUBSTITUTE SHEET (RULE 26)
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52. A ligand according to any one of claims 1 to 51, wherein the ligand
comprises a variable domain, wherein the amino acid sequences of FW1, FW2,
FW3 and FW4 are the same as the amino acid sequences of corresponding
framework regions encoded by a human germline antibody gene segment, or the
amino acid sequences of FW1, FW2 ? FW3 and FW4 collectively contain up to 10
amino acid differences relative to the amino acid sequences of corresponding
framework regions encoded by said human germline antibody gene segment.
53. The ligand according to claim 51 or claim 52, which comprises an
antibody variable domain comprising FW1, FW2 and FW3 regions, and the
amino acid sequence of said FW1, FW2 and FW3 are the same as the amino acid
sequences of corresponding framework regions encoded by human germline
antibody gene segments.
54. The ligand according to any one of claims 51 to 53, wherein said human
germline antibody gene segment is selected from the group consisting of DP47,
DP45,DP48and DPK9.
55. A ligand according to any preceding claim, comprising a Vh domain that
is not a Camelid immunoglobulin variable domain.
56. The ligand of Claim 55, comprising a Vh domain that does not contain one
or more amino acids that are specific to Camelid immunoglobulin variable
domains as compared to human Vh domains.
57. A method for producing a ligand comprising a first immunoglobulin single
variable domain having a first binding specificity and a second single
immunoglobulin single variable domain having a second binding specificity, one
or both of the binding specificities being specific for a protein which increases the
half-life of the ligand in v/vo, the method comprising the steps of:
(a) selecting a first variable domain by its ability to bind to a first epitope,
(b) selecting a second variable region by its ability to bind to a second
epitope,
(c) combining the variable regions; and
(d) selecting the ligand by its ability to bind to said first and second epitopes;
wherein, when said variable domains are complementary, neither of said domains
is a Vh domain specific for HSA.
58. A method according to claim 57 wherein said first variable domain is
selected for binding to said first epitope in absence of a complementary variable
domain.
59. A method according to claim 57 wherein said first variable domain is
selected for binding to said first epitope in the presence of a third complementary
variable domain in which said third variable domain is different from said second
variable domain.
60. Nucleic acid encoding a dual-specific ligand according to any one of
claims 1 to 56.
SUBSTITUTE SHEET (RULE 26)
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61 . A nucleic acid according to claim 60 which is specific for TNFa,
comprising the nucleic acid sequence of TAR1-5-19 or a sequence that is at least
70% homologous thereto.
62. A nucleic acid according to claim 60 which is specific for TNFa,
comprising the nucleic acid sequence of TAR1-5 or a sequence that is at least
70% homologous thereto.
63. A nucleic acid according to claim 60 which is specific for TNFa,
comprising the nucleic acid sequence of TAR1-27 or a sequence that is at least
70% homologous thereto.
64. A nucleic acid according to claim 60 which is specific for TNF receptor 1 ,
comprising the nucleic acid sequence of TAR2-10 or a sequence that is at least
70% homologous thereto.
65. A nucleic acid according to claim 60 which is specific for TNF receptor 1,
comprising the nucleic acid sequence of TAR2-10 or a sequence that is at least
80% homologous thereto.
66. A nucleic acid according to claim 60 which is specific for TNF receptor 1 5
comprising the nucleic acid sequence of TAR2h-5 or a sequence that is at least
70% homologous thereto.
67. A nucleic acid according to claim 60 which is specific for TNF receptor 1,
comprising the nucleic acid sequence of TAR2h-5 or a sequence that is at least
80% homologous thereto.
68. A nucleic acid according to claim 60 which is specific for SA, comprising
the nucleic acid sequence of MSA- 16 or a sequence that is at least 70%
homologous thereto.
69. A nucleic acid according to claim 60 which is specific for SA, comprising
the nucleic acid sequence of MSA-26 or a sequence that is at least 70%
homologous thereto.
70. A vector comprising nucleic acid according to any one of claims 60 to 69.
71 . A vector according to claim 70, further comprising components necessary
for the expression of a dual-specific ligand.
72. A host cell transfected with a vector according to claim 71 .
73 . A method for producing a closed conformation multi-specific ligand
comprising a first single epitope binding domain having a first epitope binding
specificity and a non-complementary second epitope binding domain having a
second epitope binding specificity, wherein the first and second binding
specificities are capable of competing for epitope binding such that the closed
SUBSTITUTE SHEET (RULE 26)
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140
conformation multi-specific ligand may not bind both epitopes simultaneously,
said method comprising the steps of:
a) selecting a first epitope binding domain by its ability to bind to a first
epitope,
b) selecting a second epitope binding domain by its ability to bind to a
second epitope,
c) combining the epitope binding domains such that the domains are in a
closed conformation; and
d) selecting the closed conformation multispecific ligand by its ability to bind
to said first second epitope and said second epitope, but not to both said first and
second epitopes simultaneously.
74. A method according to claim 73 wherein the first and the second epitope
binding domains are immunoglobulin variable heavy chain domains (v H )-
75. A method according to claim 73 wherein the first and the second
immunoglobulin variable domains are immunoglobulin variable light chain
domains (Vl)-
76. A method according to any one of claims 73 to 75 wherein the
immunoglobulin domains are derived from immunoglobulins directed against said
epitopes.
77. A method according to any one of claims 73 to 76, wherein said first and
second epitopes are present on separate antigens.
78. A method according to any one of claims 73 to 76, wherein said first and
second epitopes are present on the same antigen.
79. A method according to any one of claims 73 to 78 wherein the variable
domain is derived from a repertoire of single antibody domains.
80. A method of claim 79 wherein said repertoire is displayed on the surface
of filamentous bacteriophage and wherein the single antibody domains are
selected by binding of the bacteriophage repertoire to antigen.
81. A method of any one of claims 73 to 80 wherein the sequence of at least
one immunoglobulin variable domain is modified by mutation or DNA shuffling.
82. A closed conformation multispecific ligand comprising a first epitope
binding domain having a first epitope binding specificity and a non-
complementary second epitope binding domain having a second epitope binding
specificity wherein the first and second binding specificities are capable of
competing for epitope binding such that the closed conformation multi-specific
ligand cannot bind both epitopes simultaneously.
83. A closed conformation multispecific ligand according to claim 82,
obtainable by a method according to any one of claims 73 to 80.
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84. A closed conformation multispecific ligand according to claim 82 or claim
83, comprising more than one single heavy chain variable domain of an antibody
or more than one light chain variable domain of an antibody.
85. A closed conformation multi-specific ligand according to claim 84
wherein the Vh y L are linked by a peptide linker.
86. A closed conformation multi-specific ligand according to claim 84
wherein the Vh or Vl are provided by an antibody Fab-like region.
87. A closed conformation multi-specific ligand according to any one of
claims 82 to 84 wherein the variable regions are non-covalently associated.
88. A closed conformation multi-specific ligand according to any one of
claims 82 to 84 wherein the variable regions are covalently associated.
89. A closed conformation multi-specific ligand according to claim 87
wherein the covalent association is mediated by disulphide bonds.
90. A closed conformation multi-specific ligand according to any of claims 82
to 89 which comprises a universal framework.
91 . A closed conformation multi-specific ligand according to any of claims 82
to 90 which comprises a binding site for a generic ligand.
92. The closed conformation multi-specific ligand of claim 9 1 , wherein the
generic ligand binding site is selected from the group consisting of protein A,
protein L and protein G.
93. A closed conformation ligand according to any of claims 82 to 92,
wherein the ligand comprises a variable domain having one or more framework
regions comprising an amino acid sequence that is the same as the amino acid
sequence of a corresponding framework region encoded by a human germline
antibody gene segment, or the amino acid sequences of one or more of said
framework regions collectively comprises up to 5 amino acid differences relative
to the amino acid sequence of said corresponding framework region encoded by a
human germline antibody gene segment.
94. The closed conformation ligand according to claim 93, wherein the ligand
comprises a variable domain wherein the amino acid sequences of FW1, FW2,
FW3 and FW4 are the same as the amino acid sequences of corresponding
framework regions encoded by a human germline antibody gene segment, or the
amino acid sequences of FW1, FW2, FW3 and FW4 collectively contain up to 10
amino acid differences relative to the amino acid sequences of corresponding
framework regions encoded by said human germline antibody gene segment.
95. The closed conformation ligand according to claim 93 or claim 94, which
comprises an antibody variable domain comprising FW1, FW2 and FW3 regions,
SUBSTITUTE SHEET (RULE 26)
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142
and the amino acid sequence of said FW1, FW2 and FW3 are the same as the
amino acid sequences of corresponding framework regions encoded by human
germline antibody gene segments.
96. The closed conformation ligand according to any one of claims 92 to 95,
wherein said human germline antibody gene segment is selected from the group
consisting of DP47, DP45, DP48 and DPK9.
97. A closed conformation ligand according to any one of claims 92 to 96,
comprising a V H domain that is not a Camelid immunoglobulin variable domain.
98. The closed conformation ligand of Claim 97, wherein the V H domain does
not contain one or more amino acids that are specific to Camelid immunoglobulin
variable domains as compared to human Vh domains.
99. A closed conformation multi-specific ligand according to any one of
claims 82 to 98, wherein one specificity thereof is for an agent effective to
increase the half life of the ligand.
100. A kit comprising a closed conformation multi-specific ligand according to
any one of claims 82 to 99.
101 . Nucleic acid encoding at least a closed conformation multispecific ligand
according to any one of claims 82 to 99.
1 02. A vector comprising nucleic acid according to claim 101.
103. A vector according to claim 102, further comprising components
necessary for the expression of a closed conformation multispecific ligand.
1 04. A host cell transfected with a vector according to claim 1 03.
105. A method for detecting the presence of a target molecule, comprising;
(a) providing a closed conformation multispecific ligand bound to an agent, said
ligand being specific for the target molecule and the agent, wherein the agent
which is bound by the ligand leads to the generation of a detectable signal on
displacement from the ligand;
(b) exposing the closed conformation multispecific ligand to the target molecule;
and
(c) detecting the signal generated as a result of the displacement of the agent.
106. A method according to claim 1 05, wherein the agent is an enzyme, which
is inactive when bound by the closed conformation multispecific ligand.
1 07. A method according to claim 1 05, wherein the agent is the substrate for an
enzyme
1 08. A method according to claim 1 07, wherein the agent is a fluorescent,
luminescent or chromogenic molecule which is inactive or quenched when bound
by the ligand.
SUBSTITUTE SHEET (RULE 26)
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1 09. A kit for performing a method according to any one of claims 1 05-1 08,
comprising a closed conformation multispecific ligand capable of binding to a
target molecule, and optionally an agent and buffers suitable therefor.
110. A homogenous immunoassay incorporating a method according to any
one of claims 105-108.
111. A ligand according to any one of claims 1 to 56 for use in therapy.
112. A pharmaceutical composition comprising a ligand according to any one
of claims 1 to 56, and a pharmaceutically acceptable eccipient, carrier or diluent.
113. A method for preparing a chelating multimeric ligand comprising the steps
of:
(a) providing a vector comprising a nucleic acid sequence encoding a
single binding domain specific for a first epitope on a target;
(b) providing a vector encoding a repertoire comprising second binding
domains specific for a second epitope on said target, which epitope can be the
same or different to the first epitope, said second epitope being adjacent to said
first epitope; and
(c) expressing said first and second binding domains; and
(d) isolating those combinations of first and second binding domains which
combine together to produce a target-binding dimer.
114. A method according to claim 113, wherein the first and second binding
domains are associated covalently through a linker.
115. A method according to claim 113, wherein the first and second binding
domains are associated non-covalently.
116. A method according to claim 113, wherein the first and second binding
domains are associated through natural association of the domains.
117. A method according to claim 1 1 6, wherein the binding domains comprise
a Vh domain and a V K domain.
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
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t — I
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Q
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CO
on
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co an
co
s
ben
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on h3
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cn
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rd
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to
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CO
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CO
t-H
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an
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an
an
an
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>
3
to
OO LO oo
> 5^ w o
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
4/17
PCT/GB2003/002804
o
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
2*2*2*2*2
1
FIG. 4
Phage ELISA of a dual specific ScFv antibody K8
••2*2»2*2
i*2*2*2*2
»z*2*2*2*
••J:-:*:*
•I*I*2*2*
4 5
Antigens
1- HSA
2- APS
3- b-gal
4- Peanut
5- BSA
6- lysosyme
7- cytochrome c
FIG. 5
Soluble ELISA of the Dual Specific ScFv Antibody K8
0 100 200 300 400 500 600
K8 ScFv concentration (nmol)
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
5/17
PCT/GB2003/002804
Q
O
1.6
1.4
1.2
1 -
0.8-
0.6
0.4-
0.2-
0
FIG. 6
Soluble ScFv ELISA of K8V K /dummy V H clone
1
-:!:!:-:3:s:2:J;S:ir:
m m m m m m m
2 3
Antigens
4
1- BSA
2- b-gal
3- APS
4- Protein A
FIG. 7
RBS
CAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGCAAATTCTATTTCAAGGAGACAGTCATA ATG AAA TAG CTA
> M K Y L
LMB3
Sfil Ncol
TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GC G GCC CAG CCG GCC ATG GCC GAG GTG TTT
LPTAAAGLLLLA A ~ O P A M A E V F
Xhol linker
GAG TAC TGG GGC CAG GGA ACC CTG GTC ACC GT C TCG AG C GGT GGA GGC GGT TCA GGC GGA GGT
DYWGQGTLVTVS SGGGGSGGG
Sail NotI
GGC AGC GGC GGT GGC GG G TCG AC G GAC ATC CAG ATG ACC CAG GCG GCC GCA GAA CAA AAA CTC '
GSGGGGSTT) I QMTQAAAEQKL
<
link seq new
HIS -tag
CAT CAT CAT CAC CAT CAC GGG GCC GCA
HHHHHHGAA
(insertion in V domain vector 2 only
myc-tag Gene III
ATC TCA GAA GAG GAT CTG AAT GGG GCC GCA TAG ACT GTT GAA AGT TGT TTA GCA AAA CCT CAT
ISEEDLNGAA * TVESCLAKPH
<
pHEN seq
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
6/17
FIG. 8
PCT/GB2003/002804
lac RBS
promoter
■Q p15 ori J:
8
leader linker
CH vector
CH gene
{M13 ori) -
o
CD
CO
— Q:
0)32
QQ 5 Uj
myc 2x
tag ochre
Cm
FIG. 9
r
o
o
CD
CO CO
lac RBS
promoter
leader
CK vector
■ (colEI orj) -
amp
* ■ * p
CK gene
CD
O
CO
o
flag
tag
2x
ochre
<M13 ori>
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
7/17
PCT/GB2003/002804
o
o
O)
FIG. 10
TNF receptor assay
0.1 1 10 100 1000
dAb/dimer concentration nM
PEP1-5d4
PEP1-5-19d4
PEP1-519 monomer
o 120
o
o
FIG. 11
TNF receptor assay
1 10 100
dimer concentration nM
1000
PEP1-5d1
PEP1-5d2
PEP1-5d3
PEP1-5d4
PEP1-5d5
PEP1-5d6
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
8/17
PCT/GB2003/002804
FIG. 12
110
o
o
-10
TNF Receptor assay
*-PEP1-5-19 3U homodimer
PEP1-5-19 5U homodimer
PEP1-5-19 71) homodimer
*-PEP1-5-19 CH/CK homodimer
h*-PEP1-5-19 cys hinge
0.1 1 10 100 1000
dAb/dimer concentration nM
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019 PCT/GB2003/002804
9/17
FIG. 13
Dummy V H sequence for library 1
1
E
GAG
CTC
V
GTG
CAC
Q
CAG
GTC
L
CTG
GAC
L
TTG
AAC
E
GAG
CTC
S
TCT
AGA
G
GGG
CCC
G
GGA
CCT
G
GGC
CCG
L
TTG
AAC
V
GTA
CAT
Q
CAG
GTC
P
CCT
GGA
G
GGG
CCC
G
GGG
CCC
49
S
TCC
AGG
L
CTG
GAC
R
CGT
GCA
L
CTC
GAG
S
TCC
AGG
C
TGT
ACA
A
GCA
CGT
A
GCC
CGG
S
TCC
AGG
G
GGA
CCT
F
TTC
AAG
T
ACC
TGG
F
TTT
AAA
S
AGC
TCG
s
AGC
TCG
Y
TAT
ATA
97
A
GCC
CGG
M
ATG
TAC
S
AGC
TCG
W
TGG
ACC
V
GTC
CAG
R
CGC
GCG
Q
CAG
GTC
A
GCT
CGA
P
CCA
GGT
G
GGG
CCC
K
AAG
TTC
G
GGT
CCA
L
CTA
GAT
E
GAG
CTC
W
TGG
ACC
V
GTC
CAG
S
A
I
S
G
S
G
G
S
T
Y
Y
A
D
S
V
145
TCA
GCT
ATT
AGT
GGT
AGT
GGT
GGT
AGC
ACA
TAC
TAC
GCA
GAC
TCC
GTG
AGT
CGA
TAA
TCA
CCA
TCA
CCA
CCA
TCG
TGT
ATG
ATG
CGT
CTG
AGG
CAC
193
K
AAG
TTC
G
GGC
CCG
R
CGG
GCC
F
TTC
AAG
T
ACC
TGG
I
ATC
TAG
S
TCC
AGG
R
CGT
GCA
D
GAC
CTG
N
AAT
TTA
S
TCC
AGG
K
AAG
TTC
N
AAC
TTG
T
ACG
TGC
L
CTG
GAC
Y
TAT
jAT.A
241
L
CTG
GAC
Q
CAA
GTT
M
ATG
TAC
N
AAC
TTG
S
AGC
TCG
L
CTG
GAC
R
CGT
GCA
A
GCC
CGG
E
GAG
CTC
D
GAC
CTG
T
ACC
TGG
A
GCG
CGC
V
GTA
CAT
Y
TAT
ATA
Y
TAC
ATG
c
TGT
ACA
A
K
S
Y
G
A
F
D
y
W
G
Q
G
T
L
V
289
GCG
AAA
AGT
TAT
GGT
GCT
TTT
GAC
TAC
TGG
GGC
CAG
GGA
ACC
CTG
GTC
CGC
TTT
TCA
ATA
CCA
CGA
AAA
CTG
ATG
ACC
CCG
GTC
CCT
TGG
GAC
CAG
337
T
ACC
TGG
V
GTC
CAG
S
TCG
AGC
S
AGC
TCG
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
10/17
PCT/GB2003/002804
FIG. 14
Dummy V H sequence for library 2
1
E
GAG
CTC
V
GTG
CAC
Q
CAG
GTC
L
CTG
GAC
L
TTG
AAC
E
GAG
CTC
S
TCT
AGA
G
GGG
CCC
G
GGA
CCT
G
GGC
CCG
L
TTG
AAC
V
GTA
CAT
Q
CAG
GTC
P
CCT
GGA
G
GGG
CCC
G
GGG
CCC
49
S
TCC
AGG
L
CTG
GAC
R
CGT
GCA
L
CTC
GAG
S
TCC
AGG
C
TGT
ACA
A
GCA
CGT
A
GCC
CGG
S
TCC
AGG
G
GGA
CCT
F
TTC
AAG
T
ACC
TGG
F
TTT
.TVAAl
S
AGC
TCG
S
AGC
TCG
Y
TAT
ATA
97
A
GCC
CGG
M
ATG
TAC
S
AGC
TCG
W
TGG
ACC
V
GTC
CAG
R
CGC
GCG
Q
CAG
GTC
A
GCT
CGA
P .
CCA
GGT
G
GGG
CCC
K
AAG
TTC
G
GGT
CCA
L
CTA
GAT
E
GAG
CTC
W
TGG
ACC
V
GTC
CAG
S
A
I
S
G
S
G
G
S
T
Y
Y
A
D
S
V
145
TCA
GCT
ATT
AGT
GGT
AGT
GGT
GGT
AGC
ACA
TAC
TAC
GCA
GAC
TCC
GTG
AGT
CGA
TAA
TCA
CCA
TCA
CCA
CCA
TCG
TGT
ATG
ATG
CGT
CTG
AGG
CAC
193
K
AAG
TTC
G
GGC
CCG
R
CGG
GCC
F
TTC
AAG
T
ACC
TGG
I
ATC
TAG
S
TCC
AGG
R
CGT
GCA
D
GAC
CTG
N
AAT
TTA
S
TCC
AGG
K
AAG
TTC
N
AAC
TTG
T
ACG
TGC
L
CTG
GAC
Y
TAT
ATA
241
L
CTG
GAC
Q
CAA
GTT
M
ATG
TAC
N
AAC
TTG
S
AGC
TCG
L
CTG
GAC
R
CGT
GCA
A
GCC
CGG
E
GAG
CTC
D
GAC
CTG
T
ACC
TGG
A
GCG
CGC
V
GTA
CAT
Y
TAT
A.TAi
Y
TAC
ATG
C
TGT
ACA
A
K
S
y
G
A
X
X
X
X
F
D
Y
W
G
Q
289
GCG
AAA
AGT
TAT
GGT
GCT
NNK
NNK
NNK
NNK
TTT
GAC
TAC
TGG
GGC
CAG
CGC
TTT
TCA
ATA
CCA
CGA
NNK
NNK
NNK
NNK
AAA
CTG
ATG
ACC
CCG
GTC
337
•
G
GGA
CCT
T
ACC
TGG
L
CTG
GAC
V
GTC
CAG
T
ACC
TGG
V
GTC
CAG
S
TCG
AGC
S
AGC
TCG
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
11 / 17
PCT/GB2003/002804
FIG. 15
Dummy V K sequence for library 3
1
D
GAC
CTG
I
ATC
TAG
Q
CAG
GTC
M
ATG
TAC
T
ACC
TGG
Q
CAG
GTC
S
TCT
AGA
P
CCA
GGT
S
TCC
AGG
S
TCC
AGG
L
CTG
GAC
S
TCT
AGA
A
GCA
CGT
S
TCT
AGA
V
GTA
CAT
G
GGA
CCT
D
R
V
T
I
T
C
R
A
S
Q
S
I
S
S
Y
49
GAC
CGT
GTC
ACC
ATC
ACT
TGC
CGG
GCA
AGT
CAG
AGC
ATT
AGC
AGC
TAT
CTG
GCA
CAG
TGG
TAG
TGA
ACG
GCC
CGT
TCA
GTC
TCG
TAA
TCG
TCG
ATA
97
L
TTA
AAT
N
AAT
TTA
W
TGG
ACC
Y
TAC
ATG
Q
CAG
GTC
Q
CAG
GTC
K
.AA.A
TTT
P
CCA
GGT
G
GGG
CCC
K
AAA
TTT
A
GCC
CGG
P
CCT
GGA
K
AAG
TTC
L
CTC
GAG
L
CTG
GAC
I
ATC
TAG
145
Y
TAT
A
GCT
A
GCA
S
TCC
S
AGT
L
TTG
Q
CAA
S
AGT
G
GGG
V
GTC
P
CCA
S
TCA
R
CGT
F
TTC
S
AGT
G
GGC
ATA
CGA
CGT
AGG
TCA
AAC
GTT
TCA
CCC
CAG
GGT
AGT
GCA
AAG
TCA
CCG
193
S
AGT
TCA
G
GGA
CCT
S
TCT
AGA
G
GGG
ccc
T
ACA
TGT
D
GAT
CTA
F
TTC
AAG
T
ACT
TGA
L
CTC
GAG
T
ACC
TGG
I
ATC
TAG
S
AGC
TCG
S
AGT
TCA
L
CTG
GAC
Q
CAA
GTT
P
CCT
GGA
E
D
F
A
T
Y
Y
C
Q
Q
S
Y
S
T
P
N
241
GAA
GAT
TTT
GCT
ACG
TAC
TAC
TGT
CAA
CAG
AGT
TAC
AGT
ACC
CCT
AAT
CTT
CTA
AAA
CGA
TGC
ATG
ATG
ACA
GTT
GTC
TCA
ATG
TCA
TGG
GGA
TTA
289
T
ACG
TGC
F
TTC
AAG
G
GGC
CCG
Q
CAA
GTT
G
GGG
CCC
T
ACC
TGG
K
AAG
TTC
V
GTG
CAC
E
GAA
CTT
I
ATC
TAG
K
AAA
TTT
R
CGG
GCC
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
12/17
PCT/GB2003/002804
FIG. 16
Nucleotide and amino acid sequence of anti MSA dAbs MSA 16 and MSA 26
A: MSA 16
GAC
D
ATC
I
CAG
Q
ATG
M
ACC
T
CAG
Q
TCT
S
CCA
P
TCC
S
TCC
S
CTG
L
TCT
S
GCA
A
TCT
S
GTA
V
GGA
G
GAC
D
CGT
R
GTC
V
ACC
T
ATC
I
ACT
T
TGC
C
CGG
R
GCA
A
AGT
S
CAG
Q
AGC
S
ATT
I
ATT
I
AAG
K
CAT
H
TTA
L
AAG
K
TGG
w .
TAC
Y
CAG
Q
CAG
Q
AAA
K
CCA
P
GGG
G
AAA
K
GCC
A
CCT
P
AAG
K
CTC
L
CTG
L
ATC
I
TAT
Y
GGT
G
GCA
A
TCC
S
CGG
R
TTG
L
CAA
Q
AGT
S
GGG
G
GTC
V
CCA
P
TCA
S
CGT
R
TTC
F
AGT
S
GGC
G
AGT
S
GGA
G
TCT
S
G
ACA
T
GAT
D
TTC
F
ACT
T
CTC
L
ACC
T
ATC
I
AGC
S
AGT
S
CTG
L
CAA
Q
CCT
P
GAA
E
GAT
D
TTT
F
GCT
A
ACG
T
TAC
Y
TAC
Y
TGT
C
CAA
Q
CAG
Q
GGG
G
GCT
A
CGG
R
TGG
W
CCT
P
X~ "< "7\ ✓^l
CAG
Q
ACG
T
TTC
F
GGC
G
CAA
Q
GGG
G
ACC
T
AAG
K
f*n /""l
GTG
V
GAA
E
ATC
I
AAA
K
CGG
R
B: MSA 26
GAC
D
ATC
I
CAG
Q
ATG
M
ACC
T
CAG
Q
TCT
S
CCA
P
TCC
S
TCC
S
CTG
L
TCT
S
GCA
A
TCT
S
GTA
V
GGA
G
GAC
D
CGT
R
GTC
V
ACC
T
ATC
I
ACT
T
TGC
C
CGG
R
GCA
A
AGT
S
CAG
Q
AGC
S
ATT
I
TAT
Y
TAT
Y
CAT
H
TTA
L
AAG
K
TGG
W
TAC
Y
CAG
Q
CAG
Q
AAA
K
CCA
P
GGG
G
AAA
K
GCC
A
CCT
P
AAG
K
CTC
L
CTG
L
ATC
I
TAT
Y
AAG
K
GCA
A
TCC
S
ACG
T
TTG
L
CAA
Q
AGT
S
GGG
G
GTC
V
CCA
P
TCA
S
CGT
R
TTC
F
AGT
S
GGC
G
AGT
S
GGA
G
TCT
S
GGG
G
ACA
T
GAT
D
TTC
F
ACT
T
CTC
L
ACC
T
ATC
I
AGC
S
AGT
S
CTG
L
CAA
Q
CCT
P
GAA
E
GAT
D
TTT
F
GCT
A
ACG
T
TAC
Y
TAC
Y
TGT
C
CAA
Q
CAG
Q
GTT
V
CGG
R
AAG
K
GTG
V
CCT
P
CGG
R
ACG
T
TTC
F
GGC
G
CAA
Q
GGG
G
ACC
T
AAG
K
GTG
V
GAA
E
ATC
I
AAA
K
CGG
R
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
13/17
PCT/GB2003/002804
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
14/17
PCT/GB2003/002804
o
o
CO
CM
<
CO
o
s
o
o
05
CD I
o
_Q
CM
CO
CD
o>
v ~
1
i
i
i
CO
1
CO
CO
CO
I
1
1
II
ii
II
ii
o
\ >
Lo-
LL
LL
LL
• m, mm
ci
c
CO
CD
CO
CO
CM
CM
CM
CM
<D
0
CD
CD
c
o
O
o
o
o
O
o
o
CO
CO
CO
CO
o
•
O
•
o
■
o
•
■
•
•
■
1^.
CO
I
XT
II
o
CD
CM
_o
O
CO
o
i
CO
I
II
o
CO
CM
CD CD
O
O
CO
O
CO
i
II
O
LL
CO
CM
CD
C
_o
o
CO
CM
LO
CO
i
■
i
i
CO
CO
CO
CO
t
1
1
1
M
II
n
ii
II
O
o
o
LL
LL
10-
LL
m ■ ■■
c
CD
CO
CO
CO
CM
CM
CM
CM
CD
CD
CD
CD
O
O
O
O
O
O
O
O
CO
CO
CO
CO
O
■
O
■
O
■
o
■
•
•
•
1^-
•
CO
II
o
CO
CM
0)
c,
o
o
CO
o
LO
CO
o
o
CO
m
CM
CD
E
o
CM
-LO
— > o
LL. CM
i — 1 — i — 1 — i — ■ — r
o c
o
LO
CM
~i — 1 — r
o c:
LO
LO
Resp. Diff.
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
15/17
PCT/GB2003/002804
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
16/17
PCT/GB2003/002804
(a)
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
FIG. 19
dSeriesI
TAR 1 -5- TAR 1 -5- Dual
1 9dAb 1 9dAb specific
+ MSA
Dual
specific
+ MSA
(b)
biotinylated anti-TNF
TNF
chromogenic
substrate
Streptavidin-HRP
TNFRI/Fc chimera
Anti-Fc capturing antibody
TNF Receptor assay
100t
LH LH+ TNF + MSA
MSA MSA only
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019 PCT/GB2003/002804
17/17
FIG. 20
TNF Receptor assay
120 n
Serum Albumin (mg/ml)
SUBSTITUTE SHEET (RULE 26)
WO 2004/003019
1/13
PCT/GB2003/002804
CD
P
CO
LU
LU
cy
LU
CO
s
Ph
rh
CJ
CJ
cd
CD
rn
CD
CD
CD
fa fiC,
4. j
CD
CD
CD
CD
Eh
>
CJ
CD
03 CD
CJ
<
CO
CJ
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SUBSTITUTE SHEET (RULE 26)
INTERNATIONAL SEARCH REPORT
lnt< >na! Application No
PC77GB 03/02804
A. CLASSIFICATION OF SUBJECT MATTER
IPC 7 C07K16/24 C07K16/18
C12N15/63 C12N15/62
C07K 16/28
C12N15/13
A61K39/395 C07K16/46
According to International Patent Classification (IPC) orto both national classification and IPC
B. FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols)
IPC 7 C07K
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Electronic data base consulted during the international search (name of data base and, where practical, search terms used)
EPO-Internal , BIOSIS, WPI Data, PAJ, MEDLINE, EMBASE
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
A
X
Y
ELS CONRATH K ET AL: "Camel single-domain
antibodies as modular building units in
bispecific and bivalent antibody
constructs "
JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN
SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE,
MD, US,
vol . 276, no. 10,
9 March 2001 (2001-03-09), pages
7346-7350, XP002248402
ISSN: 0021-9258
cited in the application
page 7350
page 7349
-/--
1-21,
47-56,
60,111,
112
113-117
24-26,
32-35,
38,40,
47-56 ,
64-67,
70-72,
j )( I Further documents are listed in the continuation of box C.
ID
Patent family members are listed in annex.
° Special categories of cited documents :
•A" document defining the general state of the art which is not
considered to be of particular relevance
"E" earlier document but published on or after the international
filing date
"L* document which may throw doubts on priority claim(s) or
which is cited to establish the publication date of another
citation or other special reason (as specified)
"O 1 document referring to an oral disclosure, use, exhibition or
other means
"P" document published prior to the international filing date but
later than the priority date claimed
*T later document published after the international filing date
or priority date and not in conflict with the application but
cited to understand the principle or theory underlying the
invention
"X" document of particular relevance; the claimed invention
cannot be considered novel or cannot be considered to
involve an inventive step when the document is taken alone
"Y" document of particular relevance; the claimed invention
cannot be considered to involve an Inventive step when the
document is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
in the art.
document member of the same patent family
Date of the actual completion of the international search
8 June 2004
Date of mailing of the international search report
08. 07. 2004
Name and mailing address of the ISA
European Patent Office, P.B. 5818 Patentlaan 2
NL - 2280 HV Rijswijk
Tel. (+31-70) 340-2040, Tx. 31 651 epo nl,
Fax: (+31-70) 340-3016
Authorized officer
Wagner, R
Form PCT/lSA/210 (second sheet) (January 2004)
INTERNATIONAL SEARCH REPORT
Int >nal Application No
PCT7GB 03/02804
C.(Contlnuation) DOCUMENTS CONSIDERED TO BE RELEVANT
wiiaUUil <JI UUUUintJlll, Willi IllUICdllun, wnere dp|-MUJ->i idle, ui nits leiyvaiu pciij^ciytiSj
neievani to ciaim no.
111,112
Y
page 7349
27,28,
36-38,
zin
47-56,
64-67,
70-72,
111,112
page 7349
X
WO 00/29004 A (PEPT0R LTD ;PLAKSIN DANIEL
22,23,
(ID) 25 May 2000 (2000-05-25)
25,
29-31,
47-56,
61-63,
70-72,
111,112
example 2
X
REITER Y ET AL: "An antibody
22 , 23 j
single-domain phage display library of a
25,
native heavy chain variable region:
29-31,
isolation of functional single-domain VH
38,40,
molecules with a unique interface"
47-56,
UVJUl\IMr\L VJr rlULI-i'ULHIX DJ.ULUU I ? L-UIML/UN , UD,
£1 — £^
O 1 UO y
vol. 290, no. 3,
70-72,
1 January 1999 (1999-01-01), pages
111,112
685-698, XP004461990
ISSN: 0022-2836
page 687
P,X
W0 03/002609 A (MEDICAL RES COUNCIL
41,42,
;T0MLINS0N IAN (GB); WINTER GREG (GB);
82,83
IGNATOV) 9 January 2003 (2003-01-09)
page 29, line 8-15
t: A alllfJ 1 C U
P,X
W0 03/035694 A (MUYLDERMANS SERGE ; VLAAMS
22,113
INTERUNIV INST BIOTECH (BE))
1 May 2003 (2003-05-01)
page 21; example 6
Y
W0 97/30084 A (GENETICS INST)
24-26,
21 August 1997 (1997-08-21)
32-35,
/in
47-56,
64-67,
70-72,
111,112
example 6
-/--
Form PCT/ISA/210 (continuation of second sheet) (January 2004)
INTERNATIONAL SEARCH REPORT
lnt€ nal Application No
PCT/GB 03/02804
C.(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT
Category
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
x
EP 0 368 684 A (MEDICAL RES COUNCIL)
16 May 1990 (1990-05-16)
example 9
example 5
W0 98/40469 A (MCFARLAND CLIVE DAVID
;UNDERW00D PATRICIA ANNE (AU); CARDIAC CRC
N) 17 September 1998 (1998-09-17)
page 6
SMITH BRYAN J ET AL: "Prolonged in vivo
residence times of antibody fragments
associated with albumin"
BI0C0NJUGATE CHEMISTRY,
vol. 12, no. 5, September 2001 (2001-09),
pages 750-756, XP002270731
ISSN: 1043-1802
the whole document
VAN DEN BEUCKEN T ET AL: "Building novel
binding ligands to B7.1 and B7.2 based on
human antibody single variable light chain
domains"
JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB,
vol. 310, no. 3,
13 July 2001 (2001-07-13), pages 591-601,
XP004464206
ISSN: 0022-2836
abstract
-/-
39,
47-56,
111,112
24-26,
32-35,
38,40,
47-56,
64-67,
70-72,
111,112
27,28,
36-38,
40,
47-56,
64-67,
70-72,
l l 3* j l l
27,28,
36-38,
40,
47-56,
64-67,
70-72,
111,112
1-21,
47-56,
60,111,
112
1-21,
47-56,
60,111,
112
Form PCT/ISA/210 (continuation of second sheet) (January 2004)
INTERNATIONAL SEARCH REPORT
Inte na! Application No
PCT/GB 03/02804
C.(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT
Cateaorv °
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
A
MUYLDERMANS S ET AL: "UNIQUE
1-21,
i ip h B j_ - am ^m b ■ a. *ai M 1 a a ft mmwm av a^ a ■ am ■ | v. _ | * m 1 M M ■ ^™ ftk ft ™ ^™
SINGLE-DOMAIN ANTIGEN BINDING FRAGMENTS
M >>/ aa» _m
47-56,
•a* M a ft .tffete ft«a> am a a A ■ a pM | « am a V ■ % £ am am • % W m \ am *^ M ft ^% j^V H ft Ji 1 " 1
DERIVES FROM NATURALLY OCCURRING CAMEL
ft*** *X aft al al
60,111,
ft ft B|h a « 0 a m ■ ■ H M ■ ■ Ml M * aaa W rfh am. fta* f^*m « .
HEAVY-CHAIN ANTIBODIES"
a* vft a».
112
JOURNAL OF MOLECULAR RECOGNITION, HEYDEN &
\m* *a# 1 \ 1 11 ft 1— \S | II J— la Vn* Ian / III 1 \ La* »a* • m am 1 ^a» 1 J * *~~ ■ m ^"
SON LTD., LONDON, GB,
vol. 12, no. 2, March 1999 (1999-03),
4 ■ B ii i a Mm ate ate — * — aa. * * ate a.
pages 131-140, XP009012180
ISSN: 0952-3499
abstract
A
RIECHMANN L ET AL- "Sinale domain
1 \ •** la- V III If 11 11 1 La- La* I f \ Lm» * %y III \«J ■ V W l|| W*> 111
1-21
at Baa a> a
antibodies: Comparison of camel VH and
47-56,
camel ised human VH domains"
60,111,
BIOSIS,
112
XP004187632
the whole document
A
• * ^te. ft ft aa >te M ate m ^mm a M mm ate aar pa) a a >ft ar aaa 1 aa a f am »--te C*a> am f f a a
HOLLIGER P ET AL: "RETARGETING SERUM
1-21,
aa a. a a a ft * a I ate. Jte 1 ate, k s ■ ■ aa> a ■ ft ft aa aaa ft ft aa aa ate. a, ^^a ate an ^^a aa ate. a aa a av ate av %aj Ma ^te^ a pj
IMMUNOGLOBULIN WITH BISPECIFIC DIABODIES
47-56,
NATURE BIOTECHNOLOGY, NATURE PUBLISHING,
1 V » 1 1 1 V k> La* aft* %a/ 1 La> 1 111 *a* La« W VI 1 4 1 H 1 • *a/ | \ kai | W a* haa aV >✓ 1 1 aV V * %al 4
60, 111 .
US,
112
a> _* an a _» am. ate aav / >^ ate a «a a. a# "V
vol. 15, July 1997 (1997-07), pages
632-636, XP002921893
aa ate ate. a ■ - M ate ^av ate. * tew i _
ISSN: 1087-0156
abstract
A
m m ate. as .ate a a ate aw a a a—_ am va^H a ■ vte teas a ft ate. afts am. ate ate. a 1 ft a a ■ a ■ aa* aaa
US 5 644 034 A (RATHJEN DEBORAH ANN ET
22,23,
— ■ X a mmm mmm — ate. A .-^ ^- aate ate taaV a, _* X
AL) 1 Ouly 1997 (1997-07-01)
ate. aa>
25,
ate am ate —m
29-31,
38 40,
\— ' ^ft* • 1 w *
47-56,
61-63,
ftftf a- aaa ate
70-72,
111,112
column 2
■
A
■ ft aV. ate. * f a— ate ate a^a a a V am ma> am, aaa va. am aaa fta ^^ft a ft ■ a • ~aaw m — a % ft ft a^M a "V
W0 91/02078 A (PEPTIDE TECHNOLOGY LTD)
22,23,
am —m a— ■ ■ ^ft ate am. al / mm) ate a j a ate ate X
21 February 1991 (1991-02-21)
ate aa*
25, !
29-31,
38,40,
47-56,
61-63,
70-72,
111,112
page 4, line 3,19
-/~
Form PCT/ISA/210 (continuation of second sheet) (January 2004)
INTERNATIONAL SEARCH REPORT
Ini onal Application No
PCT/6B 03/02804
C.(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT
Category
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
A
P,A
TANHA J ET AL: "Optimal design features
of camel ized human single-domain antibody
1 ibraries . "
THE JOURNAL OF BIOLOGICAL CHEMISTRY.
UNITED STATES 6 JUL 2001,
vol. 276, no. 27,
6 July 2001 (2001-07-06), pages
24774-24780, XP002283749
ISSN: 0021-9258
the whole document
WO 02/072141 A (HERMAN WILLIAM)
19 September 2002 (2002-09-19)
the whole document
1-117
1-117
Form PCT/ISA/210 (continuation of second sheet) (January 2004)
INTERNATIONAL SEARCH REPORT
jrnational application No.
PCT/GB 03/02804
Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet)
This International Search Report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons:
1. L Claims Nos.:
because they relate to subject matter not required to be searched by this Authority, namely:
!. I Claims Nos.:
because they relate to parts of the International Application that do not comply with the prescribed requirements to such
an extent that no meaningful International Search can be carried out, specif icaliy:
3. I I Claims Nos.:
because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).
Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet)
This International Searching Authority found multiple inventions in this international application, as follows:
see additional sheet
1 . | Y I As all required additional search fees were timely paid by the applicant, this International Search Report covers all
L-^-J searchable claims.
2. | | As all searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment
of any additional fee.
3. I I As only some of the required additional search fees were timely paid by the applicant, this International Search Report
1 ' covers only those claims for which fees were paid, specifically claims Nos.:
4. | | No required additional search fees were timely paid by the applicant. Consequently, this International Search Report is
restricted to the invention first mentioned in the claims; it is covered by claims Nos.:
Remark on Protest [ [ The additional search fees were accompanied by the applicant's protest.
[ X | No protest accompanied the payment of additional search fees.
Form PCT/ISA/210 (continuation of first sheet (1)) (July 1998)
International Application No. PCT/ GB 03/02804
FURTHER INFORMATION CONTINUED FROM PCT/ISA/ 210
This International Searching Authority found multiple (groups of)
inventions in this international application, as follows:
1. Claims: 1-7,8-10,11-21,47-56 (all 10 in part),
60 (in part), 111-112 (both in part)
Ligand comprising a first immunoglobulin variable domain
having a first antigen or epitope specificity and a second
immunoglobulin variable domain having a second antigen or
epitope specificity wherein one or both of the antigens or
epitopes acts to increase the half-life of the ligand in
vivo, and the variable domains are not complementary to one
another.
2. Claims: 22,23,25,29-31, 38 (in part), 40,
47-56 (all 10 part), 61-63, 70-72 (all 3 in part),
111-112 (both in part)
Single domain antibody monomer ligand specific for TNF- .
3. Claims: 24,26,25,32-35, 38 (in part), 40,
47-56 (all 10 in part), 64-67,
70-72 (all 3 in part), 111-112 (both in part)
Single domain antibody monomer ligand specific for TNF
receptor 1.
4. Claims: 27, 28, 36,37, 38 (in part)40,
47-56 (all 10 in part), 64-67,
70-72 (all 3 in part), 111-112 (both in part)
Single domain antibody monomer ligand specific for serum
albumin.
5. Claims: 39, 47-56 (all 10 in part),
111-112 (both in part).
Single domain antibody monomer ligand comprising a terminal
Cys residue.
6. Claims: 41-46, 47-56 (all 10 in part), 60 (in part).
A dual specific ligand comprising at least a single domain
antibody monomer according to inventions 2-5.
page 1 of 2
International Application No. PC77 GB 03/02804
FURTHER INFORMATION CONTINUED FROM PCT/ISA/ 210
7. Claims: 73-81, 82-104, 105-110
A closed conformation multi specif ic ligand comprising a
first epitope binding domain having a first epitope binding
specificity and a non-complementary second epitope binding
domain having a second epitope binding specificity, wherein
the first and second binding specificities are capable of
competing for epitope binding such that the closed
conformation multi-specific ligand cannot bind both
epitopes simultaneously. Methods for producing the closed
conformation multispecif ic ligand. Methods using the closed
conformation multispecif ic ligands.
8. Claims: 113-117
Method for preparing a chelating multimeric ligand, specific
for adjacent epitopes.
page 2 of 2
INTERNATIONAL SEARCH REPORT
In ional Application No
PCT/GB 03/02804
Patent document
Publication
Patent family
Publication
cited in search report
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25-05-2000
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WO 02072141 A W0 03057732 A2 17-07-2003
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